Dipartimento di Biochimica e Biotecnologie Mediche e di,
Medicina Clinica e Sperimentale,
Università di Napoli "Federico II",
Napoli, Italy,
IRCCS-CSS,
S. Giovanni Rotondo, Italy
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.
References
Department of Clinical Biochemistry,
Herlev University Hospital,
Herlev, Denmark
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
studies1 2 3 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 men1 and more than 10 000
women and men2 3 and included
prospective,1 retrospective, cross-sectional, and
case-referent/case-control designs,2 3 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.4 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
studies5 6 7 8 9 and may therefore be a marker for
some other disease manifestation associated with plasma ACE
levels.
References
© 1998 American Heart Association, Inc.
Correspondence
A Fast and Accurate Method for Genotyping the Angiotensin-Converting Enzyme I/D Polymorphism
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