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Technical Briefs |
1 Institute of Clinical Pharmacology,2 Sleep Medical Centre, and4 Department of Internal Medicine, Charité, Humboldt University of Berlin, Berlin, Germany;3 Institute of Pharmacology, Ernst Moritz Arndt University, Greifswald, Germany;5 TIB MOLBIOL, Syntheselabor, Berlin, Germany;6 Epidauros Biotechnology AG, Bernried, Germany
aaddress correspondence to this author at: Institute of Clinical Pharmacology, University Hospital Charité, Campus Charité Mitte, Schumannstrasse 20/21, 10117 Berlin, Germany; fax 49-30-450-525932, e-mail christian.meisel{at}charite.de
Of the three endothelin peptides, endothelin-1, -2, and -3, endothelin-1 (EDN1) is the predominant isoform in the vascular system. EDN1 is a potent endogenous vasoconstrictor, has positive inotropic and chronotropic effects and mitogenic properties, influences homeostasis, and stimulates the renin-angiotensin-aldosterone and the sympathetic systems (1)(2)(3). EDN1 plays an important role in the cardiovascular system (4).
Pre-pro-EDN1 mRNA (2026 nucleotides) is the product of the human EDN1 gene (6836 nucleotides), which is located on chromosome 6p23-p24. The mature 21-amino acid EDN1 is generated by subsequent enzymatic cleavage of the big-EDN1 (1). Eight variants of EDN1, which may influence the hereditary risk of cardiovascular diseases, including coronary heart disease, hypertension, and ventricular arrhythmia (5)(6)(7)(8)(9)(10)(11)(12), have already been located and examined:
By sequencing the EDN1 gene of 56 unrelated Caucasians systematically, we identified four novel common genetic variations:
Genotyping methods for large numbers of samples have been reported for only 3 of the 12 partly novel polymorphisms (G+356/in4, +138/ex1ins/delA, and Lys198Asn) (5)(6)(7)(9)(11)(12). We therefore established standard PCR-restriction fragment length polymorphism (RFLP) methods for the 12 polymorphisms listed above and a melting peak analysis method using fluorescent probes (LightCycler assays) for the rapid detection of possibly functionally important EDN1 polymorphisms [except for +138/ex1ins/delA, for which a TaqMan assay already exists in our laboratory (11)]. To minimize assay costs, methods were optimized for minimal amounts of hybridization probes and restriction enzymes.
High-molecular-weight DNA from 300 healthy Caucasian volunteers was extracted from EDTA-blood with use of the MagnaPur DNA-Isolation Kit (Roche Diagnostics). Primers and restriction enzymes for PCR-RFLP methods and primers and hybridization probes for melting peak analysis (see the Data Supplement accompanying the online version of this Technical Brief at http://www.clinchem.org/content/vol50/issue2/) were chosen according to the EDN1 sequence (OMIM*131240; GenBank accession no. J05008), using Oligo Primer Analysis Software 6.0 and LightCycler Probe Design Software.
In some PCR-RFLP assays (G-1396A, +138/ex1ins/delA, and T+30/in2G; see the online Data Supplement), primer mismatches were incorporated to create recognition sites for restriction enzymes. One of the PCR-RFLP assays was established for simultaneous detection of two polymorphisms (T+834/ex5C and T+881/ex5C). All PCRs were performed in a total volume of 20 µL. The reaction mixture contained 0.2 µM each of the primers (TIB MOLBIOL), 0.1 mM each of the deoxynucleotide triphosphates (BioTherm), 2 µL of 10x Taq Buffer (BioTherm), 1 µL of the sample (containing 30–50 ng/µL genomic DNA), 1 U of Taq Polymerase (BioTherm), and assay-specific amounts of MgCl2 (1.2–2.0 mM; see the online Data Supplement). The PCR was performed in a Thermocycler (GeneAmp PCR System 9700; Perkin-Elmer) with the following conditions: initial denaturation at 95 °C for 120 s, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at assay-specific temperatures for 30 s (see the online Data Supplement), extension at 72 °C for 60 s, and a final extension at 72 °C for 7 min. For RFLP analysis, a 20-µL aliquot containing 10 µL of the PCR product was digested with 1 (T+30/in2G), 2 (+138/ex1ins/delA), 5 (G+356/in4, T+834/ex5C, and T+881/ex5C), 6 (G-1396A), or 10 U (G-46/in1A) of assay-specific restriction enzymes (MBI Fermentas GmbH; see the online Data Supplement) at the enzyme-specific temperatures overnight. Digestion products (see the online Data Supplement) were visualized by SYBR Green staining after electrophoresis in 3% agarose gels.
Hybridization probes for LightCycler assays were labeled with LightCycler Red640 or Red705. One hybridization probe pair could be used to detect two polymorphisms simultaneously (Lys198Asn and Glu106Glu). PCR was carried out in glass capillaries in a total volume of 20 µL. The reaction mixture contained assay-specific amounts of each primer (0.2–0.25 µM; see the online Data Supplement), anchor and sensor (0.05–0.1 µM; see the online Data Supplement), 0.1 mM each of the deoxynucleotide triphosphates; 30 mg/L bovine serum albumin, 50 mL/L dimethyl sulfoxide, 2 µL of 10x Taq Buffer, 1 µL of the sample, 1 U of Taq polymerase, and assay-specific amounts of MgCl2 (2.0–3.0 mM; see the online Data Supplement).
Conditions were optimized as follows: Conditions for the denaturation steps and extension temperatures were always the same. For the T-1370G PCR, the initial denaturation was at 95 °C for 120 s, followed by 30 cycles of denaturation for 0 s at 95 °C, annealing for 20 s at 53 °C, and extension for 40 s at 72 °C. For melting curve analysis, the conditions were 20 s of denaturation at 95 °C and 20 s of annealing at 32 °C, after which the temperature was continuously increased up to 70 °C (ramp rate, 0.15 °C/s).
For the T-37/in2C PCR, the initial denaturation was followed by 35 cycles of denaturation for 10 s, annealing at 58 °C, and 20 s of extension. The conditions for melting curve analysis were denaturation followed by annealing for 20 s at 40 °C, after which the temperature was continuously increased up to 80 °C (ramp rate, 0.2 °C/s).
For the -38/in4ins/delT PCR, initial denaturation was followed by 35 cycles of denaturation for 10 s, annealing at 58 °C, extension for 25 s. The conditions for melting curve analysis were denaturation followed by 20 s of annealing at 35 °C, after which the temperature was continuously increased up to 65 °C (ramp rate, 0.1 °C/s).
For the PCR for Lys198Asn and Glu106Glu, initial denaturation was followed by 43 cycles of denaturation, 20 s of annealing at 60 °C, and extension for 30 s. For melting curve analysis, denaturation was followed by 20 s of annealing at 49 °C, after which the temperature was continuously increased up to 75 °C (ramp rate, 0.1 °C/s).
Fluorescence was recorded during the heating. The melting curves (F/T) were converted to melting peaks (-dF/dT); their analysis showed distinct temperatures for all polymorphisms (see Fig. 1
and the online Data Supplement).
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Genotyping results obtained by PCR-RFLP analysis and by melting peak analysis with fluorescent probes correlated perfectly with DNA sequencing (PrismTM 310 Genetic Analyzer; Applied Biosystems).
We have established reliable and cost-efficient methods for the detection of 12 EDN1 polymorphisms. The melting peak analysis with fluorescent probes allows rapid detection of the common functional polymorphisms. High-throughput analysis can be performed by transferring the assays (using the same primers and hybridization probes) to a LightTyper instrument.
Acknowledgments
This work was supported by the German Federal Ministry of Education and Research (Grants 01 GG 9845/5, 031U209B, and NBL 3-3.1.3). We thank Anja Alfandega for technical assistance.
References
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