A Fast and Accurate Method for Genotyping the Angiotensin-Converting Enzyme I/D Polymorphism
To the Editor:
The association of the insertion/deletion (I/D) polymorphism of the ACE gene with ischemic vascular disease is disputed.1 Mistyping as DD of 4% to 5% of ID subjects may contribute to conflicting findings.2 3 The original method4 preferentially extends allele D. The addition of 5% DMSO, or the use of an allele-specific oligonucleotide (ASO), reduces mistyping.5 6 Although the ASO technique is the method of choice,2 3 5 it is time consuming because a second nested PCR must be performed on the DD samples obtained with the original method. We have devised a one-step multiplex PCR, which we tested on 520 individuals and compared the results with those obtained with other methods.
An insertion-specific primer pair (hace5a, hace5c)2 and a sense primer flanking the Alu-type sequence4 are used simultaneously. One hundred micrograms of genomic DNA are amplified on a PTC100 thermal cycler (MJ Research) in a total volume of 20 μL containing 10, 2.5, and 15 picomoles of the Alu-flanking sense primer, the insertion-specific primer, and the antisense primer, respectively, 1.5 mmol/L MgCl2, 50 μmol/L of each dNTP and 0.5 U of Taq polymerase (Promega). After 40-second denaturation at 94°C, we ran 32 cycles of 30 seconds at 94°C, 45 seconds at 68°C, and 90 seconds at 72°C, followed by 3 minutes at 72°C. The products are identified according to standard procedures.7 The D allele generates a 234-bp band; allele I produces two bands of 522 and 335 bp, respectively. Similar to the ASO-PCR protocol, the multiplex PCR detected 7 samples out of 274 ID individuals that were mistyped as DD with the original procedure (2.6%). The DMSO protocol showed only three misclassifications (1.1%) in this setting. Thus this fast, reliable new PCR procedure is an improvement in the detection of the ACE gene I/D polymorphism.
- Copyright © 1998 by American Heart Association
Agerholm-Larsen B, Nordestgaard BG, Steffensen R, Sørensen TIA, Jensen G, Tybjærg-Hansen A. ACE gene polymorphism: ischemic heart disease and longevity in 10150 individuals: a case-referent and retrospective cohort study based on the Copenhagen city heart study. Circulation. 1997;95:2358–2367.
Rigat B, Hubert C, Corvol P, Soubrier F. PCR detection of the insertion/deletion polymorphism of the human angiotensin converting enzyme gene (DCP1) (dipeptidyl carboxypeptidase 1). Nucleic Acids Res. 1992;20:1433.
Dr Mancini and coworkers describe a new, multiplex PCR procedure for fast detection of the ACE gene insertion/deletion (I/D) polymorphism but do not come to a decision as to the possible application of ACE I/D genotyping. Although mistyping of 4% to 5% of ID subjects as DD may have contributed to conflicting results of the ACE gene polymorphism on risk of ischemic heart disease in some of the early studies that did not correct for mistyping, this is unlikely for the three largest studiesR1 R2 R3 because they all corrected for mistyping by reanalyzing all DD individuals in a PCR with an insertion-specific primer. In these studies that comprised approximately 3600 menR1 and more than 10 000 women and menR2 R3 and included prospective,R1 retrospective, cross-sectional, and case-referent/case-control designs,R2 R3 the ACE I/D polymorphism was not associated with risk of ischemic heart disease, coronary artery stenosis, myocardial infarction, ischemic cerebrovascular disease, or longevity. Furthermore, a recent meta-analysis including 15 studies comprising almost 9000 individuals suggested publication bias toward positive results for the smaller studies.R4 In our view, therefore, the ACE I/D polymorphism is not suitable as a marker for ischemic heart disease, coronary artery stenosis, myocardial infarction, ischemic cerebrovascular disease, or related diagnoses and should not be used as such. However, the ACE polymorphism D-allele has consistently been associated with elevated plasma ACE levels and activity in a codominant pattern in most studiesR5 R6 R7 R8 R9 and may therefore be a marker for some other disease manifestation associated with plasma ACE levels.
Lindpaintner K, Pfeffer MA, Kreutz R, Stampfer MJ, Grodstein F, LaMotte F, Buring J, Hennekens CH. A prospective evaluation of an angiotensin-converting-enzyme gene polymorphism and the risk of ischemic heart disease. N Engl J Med. 1995;332:706–711.
Agerholm-Larsen B, Nordestgaard BG, Steffensen R, Sørensen TIA, Jensen G, Tybjærg-Hansen A. ACE gene polymorphism: ischemic heart disease and longevity in 10150 individuals: a case-referent and retrospective cohort study based on the Copenhagen City Heart Study. Circulation. 1997;95:2358–2367.
Samani NJ, Thompson JR, O’Toole L, Channer K, Woods KL. A meta-analysis of the association of the deletion allele of the angiotensin-converting enzyme gene with myocardial infarction. Circulation. 1996;94:708–712.
Rigat B, Hubert C, Alhenc-Gelas F, Cambien F, Corvol P, Soubrier F. An insertion/deletion polymorphism in the angiotensin I-converting enzyme gene accounting for half the variance of serum enzyme levels. J Clin Invest. 1990;86:1343–1346.
Lachurié M-L, Azizi M, Guyene T-T, Alhenc-Gelas F, Ménard J. Angiotensin-converting enzyme gene polymorphism has no influence on the circulating renin-angiotensin-aldosterone system or blood pressure in normotensive subjects. Circulation. 1995;91:2933–2942.
Nakai K, Itoh C, Miura Y, Hotta K, Musha T, Itoh T, Miyakawa T, Iwasaki R, Hiramori K. Deletion polymorphism of the angiotensin I-converting enzyme gene is associated with serum ACE concentration and increased risk for CAD in the Japanese. Circulation. 1994;90:2199–2202.
Winkelmann BR, Nauck M, Klein B, Russ AP, Böhm BO, Siekmeier R, Ihnken K, Verho M, Gross W, März W. Deletion polymorphism of the angiotensin I-converting enzyme is associated with increased plasma angiotensin-converting enzyme activity but not with increased risk for myocardial infarction and coronary artery disease. Ann Intern Med. 1996;125:19–25.