From the Cardiovascular Division, Department of Medicine, Brigham and
Women's Hospital (R.Y.L.Z., V.S.R., K.L.), and the Department of
Cardiology, Children's Hospital, Harvard Medical School (K.L.), Boston,
Mass; the Dean Medical Center, Oregon, Wisc (R.Z.P.); the Merck Research
Laboratories, West Point, Pa (C.S.S.); the Max Delbruck Centre for Molecular
Medicine, Berlin, Germany (K.L.); and the Cardiovascular Discovery Unit, F.
Hoffmann-La Roche Ltd, Basel, Switzerland (K.L.).
Correspondence to Klaus Lindpaintner, MD, Cardiovascular Division, Brigham and Women's Hospital, 75 Francis St, Boston, MA 02115. E-mail kl{at}calvin.bwh.harvard.edu
A prime candidate for a protein that might interact differentially with
ACEIs on the basis of genetic variation is, of course, ACE
itself. Thus, the I/D polymorphism of ACE has
recently been proposed as possibly being involved in ACEI-induced
cough,4 5 6 a speculation fueled by the
observation that the incidence of cough among patients under ACEI
treatment is roughly similar to the proportion of the population
that is homozygous for the ACE insertional allele (about
15% to 20%).
More recently, a possible link between ACEI treatment–associated
adverse effects and chymase has also been suggested: as chymase
represents an alternative pathway for the activation of
angiotensin II, it is possible that ACE inhibition may lead
to an increased biological significance of this enzyme. Release of
chymase from mast cells results in a range of profound proinflammatory
changes; in the dermis, this is associated with skin rashes, whereas in
the lungs bronchial and pulmonary infiltration with
inflammatory cells and altered regulation of vasoactive
peptides7 8 are observed. A recent report
describing an association between a polymorphism of the gene
encoding MCC and atopic eczema in a Japanese
population,7 along with the fact that skin rashes
are another adverse effect of ACEIs, therefore raises the possibility
that such molecular variants of this enzyme may also play a role in
ACEI-related cough.
Even more recently, a possible link between ACEI
treatment– associated adverse effects and B2-bradykinin
receptor has also been suggested: since B2-bradykinin receptor mediates
most of the inflammatory actions of bradykinin and is widely
present in most tissues,9 10 a genetic defect
of B2BKR may lead to altered biological activities of the
functional protein. A recent report implicating an association of an
exon 1 polymorphism on B2BKR and bradykinin-induced
angioedema in patients with C1-inhibitor
deficiency,10 as well as with the fact that
angioedema is yet another side effect of ACEIs, strongly raises the
possible involvement of B2BKR genotype in the
underlying ACEI-induced cough mechanism and the clinical manifestations
associated with ACEI treatment.
It was therefore of interest to test whether genetic polymorphisms
of these three enzymes may be associated with incidence of ACEI-related
cough. DNA samples collected in the course of a very carefully designed
and executed clinical study allowed us to pursue this goal.
ACE D/I Genotype
Determination
MCC BstXI Genotype
Determination
B2BKR +/- Genotype Determination
To confirm genotype assignment, the PCR-RFLP procedure was
performed on all samples on two separate occasions. PCR results were
scored blinded as to case-control status.
Statistical Analysis
ACE D/I
Polymorphism
MCC BstXI Polymorphism
B2BKR +/- Polymorphism
Among the strengths of our study is the very careful design which,
based on positive response to rechallenge with drug and on negative
response to placebo, removes as much as possible a major factor of
uncertainty (namely, the specificity of the ACEI causation of cough)
from the investigation and therefore adds power to the study. On the
other hand, the stringent criteria required for inclusion into the
study also resulted in a sample of only modest size. Given the number
of subjects available, therefore, we reach 80% power in the additive
models to rule out ORs of 1.87, 1.87, and 1.94 for
ACE, MCC, and B2BKR, respectively; for
dominant or recessive models, power is less. Thus, we can be confident
that by measure of the association of the polymorphisms tested,
none of the three genes contributes materially to the cough
phenotype in this sample, which was highly selected to allow
recognition of true pathogenic factors over random noise. The
observation that allele frequencies for ACE and
B2BKR variants are in agreement with previously reported
data in white subjects10 12 provides additional
support for the validity of our study.
It is important to recognize that association studies like the
present one only examine the possible association between
phenotype and the actually tested polymorphism; such
studies cannot exclude that examination of a different polymorphism
(which would of course by definition have to be in linkage
disequilibrium with the one tested) might obtain different results.
The negative results regarding the MCC and B2BKR
polymorphisms signal—within the constraints of statistical
power—that these genes (or at least the particular polymorphisms
studied) do not play an important role in ACEI-related cough in our
study population. However, because the original observation of an
association between the MCC marker and atopic conditions was
made in Japanese subjects, and the B2BKR variant and
bradykinin-induced angioedema was made in patients with
C1-inhibitor deficiency, it is possible that racial/ethnic
differences as well as selection of disease phenotypes may also
account for our data. In addition, of course, the hypothesis that there
may be a common denominator for the susceptibility to atopic skin
rashes, to C1-inhibitor deficiency–associated angioedema,
and to ACEI-related cough is a speculative one.
Because expression of ACEI-related dry cough likely represents
a multifactorial phenomenon that involves a complex interaction of
multiple interrelated factors, the possibility of the involvement of
other genes that might be responsible for the proinflammatory cascade
of actions exists, and larger studies will be needed to further explore
the possibility of genetic predisposition in ACEI-related dry
cough.
In conclusion, we found no association of the three polymorphisms
tested with ACEI-related dry cough in a highly selected group of
subjects. However, the present study certainly presents one of
the potential applications in defining pharmacogenetics in drug therapy
and will attract more attention to better understanding of drug design,
as well as development, in the near future.
Received September 30, 1997;
first decision October 29, 1997;
accepted November 25, 1997.
2.
Fuller RW, Choudry NB. Increased cough reflex
associated with angiotensin-converting enzyme
inhibitor cough. Br Med J. 1987;295:1025–1026.
3.
Bucker CE, Neilly JB, Carter R, Stevenson RD, Semple
PF. Bronchial hypersensitivity in patients who cough after receiving
angiotensin-converting enzyme inhibitors.
Br Med J. 1988;296:880–888.
4.
Yeo WW, Ramsey LE, Morice AH. ACE
inhibitor cough: a genetic link. Lancet. 1991;337:187. Letter.
5.
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.
6.
Tiret L, Rigat B, Visvikis S, Breda C, Corvol P,
Cambien F, Soubrier F. Evidence, from combined segregation and linkage
analysis, that a variant of the angiotensin
I-converting enzyme (ACE) gene controls plasma ACE levels.
Am J Hum Genet. 1992;51:197–205.[Medline]
[Order article via Infotrieve]
7.
Mao XQ, Shirakawa T, Yoshikawa T, Yoshikawa K, Kawai
M, Sasaki S, Enomoto T, Hashimoto T, Furuyama J, Hopkin JM, Morimoto K.
Association between genetic variants of mast-cell chymase and eczema.
Lancet. 1996;348:581–583.[Medline]
[Order article via Infotrieve]
8.
Caughey GH, Zerweck EH, Vanderslice P. Structure,
chromosomal assignment, and deduced amino acid sequence of a human gene
for mast cell chymase. J Biol Chem. 1991;266:12956–12963.
9.
Braun A, Kammerer S, Bohme E, Muller B, Roscher AA.
Identification of polymorphic sites of the human bradykinin B2
receptor gene. Biochem Biophys Res Commun. 1995;211:234–240.[Medline]
[Order article via Infotrieve]
10.
Lung CC, Chan ED, Zuraw BL. Analysis of an exon
1 polymorphism of the B2 bradykinin receptor gene and its
transcript in normal subjects and patients with C1
inhibitor deficiency. J Allergy Clin Immunol. 1997;99:134–146.[Medline]
[Order article via Infotrieve]
11.
Lacourciere Y, Lefebvre J, Nakhle G, Faison EP, Snavely
DB, Nelson EB. Association between cough and
angiotensin-converting enzyme inhibitors versus
angiotensin II antagonists: the design of a
prospective, controlled study. J Hypertens.
1994;12(suppl 2):49–53.
12.
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. 1993;332:706–711.
© 1998 American Heart Association, Inc.
Scientific Contributions
Three Candidate Genes and Angiotensin-Converting Enzyme Inhibitor–Related Cough
A Pharmacogenetic Analysis
Abstract
Top
Abstract
Introduction
Methods
Results
Discussion
References
Abstract—Unexplained, persistent
cough limits the use of angiotensin-converting enzyme (ACE)
inhibitors in a significant number of patients. It has been
speculated that occurrence of this adverse effect is genetically
predetermined; in particular, variants of the genes encoding ACE,
chymase, and B2-bradykinin receptor have been implicated. To
investigate this question, we determined genotypes for common
polymorphisms for these three genes in subjects with a history of
ACE inhibitor–related cough. Specificity of the adverse
effect was confirmed by a blinded, double-crossover design protocol in
which subjects were rechallenged with either lisinopril or
placebo. In 99 case subjects and 70 control subjects (who failed to
develop cough on rechallenge with ACE inhibitor) thus
selected, frequencies for the ACE D and I
alleles were 0.56 and 0.44 (cases) and 0.56 and 0.44 (controls),
respectively; frequencies for chymase A and
B alleles (absence/presence of BstXI
site) were 0.56 and 0.44 (cases) and 0.46 and 0.54 (controls),
respectively; frequencies for B2-bradykinin receptor +
and - alleles (presence/absence of a 21 to 29
nonanucleotide sequence) were 0.52 and 0.48 (cases) and
0.53 and 0.47 (controls), respectively. All observed genotype
frequencies were in Hardy-Weinberg equilibrium. There was no evidence
for association between genotype at either gene examined and
cough (adjusted for gender and age). Our data indicate that common
genetic variants of ACE, chymase, and B2-bradykinin receptor do not
explain the occurrence of ACE inhibitor–related cough.
Key Words: angiotensin-converting enzyme inhibitors • cough • angiotensin-converting enzyme • chymase • receptors, bradykinin • polymorphism • genetics
Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
References
Angiotensin-converting
enzyme inhibitors are widely used for the treatment of
hypertension and are presently the uncontested drugs of choice for
the treatment of congestive heart failure.1 The
major adverse effect encountered with ACEI treatment, and the most
frequent reason for withdrawal of the drug, is a persistent, dry
(nonproductive) cough.2 3 So far, no specific
risk factors or patient characteristics that would predict the
occurrence of this cough have been identified, and the underlying
mechanism remains unclear; however, it has been speculated that the
kininogen-kinin (bradykinin) system may be involved: inhibition of ACE
may result in a local accumulation of bradykinin, which in turn may
lead to the activation of proinflammatory peptides (eg, substance P,
phospholipase C and/or A2,
prostaglandins, neuropeptide Y) and to local release of
histamine in the airways.2 3 Because cough is a
class effect of ACEIs, and because its occurrence is not predicted by
any external factors, it seems reasonable to suspect that a primary,
genetically determined characteristic resulting in alteration of drug
action or drug metabolism may be
responsible.4
Methods
Top
Abstract
Introduction
Methods
Results
Discussion
References
Subject Selection
Our study was based on the population entered into a previously
described international multicenter trial that was set up to examine
the association of cough with ACEI and with AT1
receptor blocker treatment, respectively.11 In
brief, individuals who fulfilled the inclusion criteria (history of
ACEI-associated cough that resolved when not taking medication)
underwent a double-blinded, placebo-controlled crossover challenge with
the ACEI lisinopril at a daily dose of 20 mg, for up to 6
weeks. Ninety-nine case subjects who developed cough while taking
lisinopril, but not while taking placebo, were thus
identified. Seventy individuals who did not develop cough during
lisinopril challenge were classified as control
subjects.
The details of ACE D/I genotype determination
have previously been described.12 In brief, the
D and I alleles were identified on the basis
of PCR amplification of the respective fragments from intron 16 of
ACE and by subsequent electrophoretic size fractionation and
ethidium bromide visualization. Because the D allele in
heterozygotes is preferentially amplified, all DD
genotype samples were subjected to a second independent PCR
amplification with a primer pair that recognizes an insertion-specific
sequence to ensure accurate genotyping.
Genotyping for the BstXI polymorphism, a G3255A
variant in a region of the 5'-flanking sequence, involved a PCR-RFLP
technique. The assay was carried out in whole blood with the use of
Gene Releaser (Bioventures) according to the manufacturer's
recommendations. Reagent concentrations in the 15-mL PCR reaction were
330 nmol/L each for sense (5'-TGC CCC ACA TCA ACA TTC ATT C-3') and
reverse (5'-TCC GGA GCT GGA GAA CTC TTG T-3') primers, 166 mmol/L
deoxynucleotide triphosphates, 2.5 mmol/L
MgCl2, and 0.15 U of Taq polymerase.
Samples were amplified for 36 cycles consisting of denaturation at
94°C for 30 seconds, annealing at 56°C for 45 seconds, and
extension at 72°C for 1 minute, followed by a final extension step at
72°C of 5 minutes. The resulting 645-bp PCR fragment was incubated at
55°C for 7 hours with 3 U of the restriction endonuclease
BstXI (New England Biolabs), according to the
manufacturer's specification, and restriction fragments were
identified on 2% ethidium bromide–stained agarose gels. Genotypic
polymorphisms of MCC were defined as AA
(homozygous for absence of restriction site), BB (homozygous
for presence of restriction site), or AB
(heterozygous).7
Genotyping for the presence/absence of a 9-bp sequence, a 21 to
29 nonanucleotide variant (5'-GGTGGTGAC-3') in a region of
the 5'-untranslated sequence,10 involved a
radioactive-labeling/PCR technique. The assay was carried out in whole
blood with the use of GeneReleaser (Bioventures) according to the
manufacturer's recommendations. Reagent concentrations in the 15-mL
PCR reaction were 330 nmol/L each for sense (5'-CTG TTC CCG CCG CCA CTC
CA-3') and reverse (5'-CAG AGG TGA GGC GGC TGG AG-3')
primers,10 166 mmol/L
deoxynucleotide triphosphates, 3.0 mmol/L
MgCl2, and 0.15 U of Taq polymerase.
The sense primer was labeled at the 5' end with
[32P]
-ATP. Samples were amplified for 36
cycles consisting of denaturation at 94°C for 30 seconds, annealing
at 62°C for 45 seconds, and extension at 72°C for 1 minute,
followed by a final extension step at 72°C of 5 minutes. The reaction
products were resolved over denaturing sequencing gels containing
6% polyacrylamide, 8 mol/L urea, and 30% formamide and
visualized by autoradiography. Genotypic
polymorphisms of B2BKR were defined as ++ (homozygous
for presence of nonanucleotide sequence), -- (homozygous
for absence of nonanucleotide sequence), or
+-(heterozygous).10
Alleles and genotype frequencies among case and
control subjects were counted and compared by
2 test with Hardy-Weinberg predictions.
In addition, multivariate regression analysis
was carried out, adjusting for age and gender, to determine the odds
ratio for each genotype to predict the occurrence of cough.
Data for each polymorphism were analyzed under assumptions
of an additive, dominant, and a recessive model, respectively, using a
two-tailed value of P<.05 as the criterion for statistical
significance.
Results
Top
Abstract
Introduction
Methods
Results
Discussion
References
Study Sample Characteristics
There were 47 men and 52 women among the case subjects and 24 men
and 46 women among the control subjects. Mean±SD ages of case and
control subjects were not different (57.0±10.1 and 58.4±10.1 years,
respectively; P=.37).
Allele frequencies for D and I
alleles were 0.56 and 0.44 in case subjects and 0.56 and 0.44 in
control subjects, respectively. Genotype frequencies were 0.232
for II, 0.414 for ID, and 0.354 for DD
in cases; 0.171 for II, 0.529 for ID, and 0.300
for DD in controls; and 0.226 for II, 0.445 for
ID, and 0.329 for DD in both groups combined.
Genotype frequencies did not deviate from the Hardy-Weinberg
equilibrium in controls
(22df=0.30,
P=.86), cases
(22df=1.33,
P=.51), or the whole study group
(22df=0.30,
P=.86). No overall difference in genotype
distribution was seen among cases and controls
(22df=2.25,
P=.32). Logistic regression analysis assuming
additive (DD versus DI versus II),
dominant (DD and DI versus II), or
recessive (DD versus DI and II)
inheritance likewise failed to reveal any significant association
between phenotype and genotype, with ORs (95% CIs) of
0.98 (0.64 to 1.50), 0.69 (0.32 to 1.52), and 1.28 (0.66 to 2.46),
respectively.
Allele frequencies for A and B
alleles were 0.56 and 0.44 in case subjects and 0.46 and 0.54 in
control subjects, respectively. Genotype frequencies were 0.333
for AA, 0.444 for AB, and 0.222 for
BB, respectively, in case subjects; and 0.196 for
AA, 0.518 for AB, and 0.286 for BB,
respectively, in control subjects. Again, genotype frequencies
did not deviate from the Hardy-Weinberg equilibrium in controls
(22df=0.061,
P=.97), cases
(22df=0.55,
P=.76), or the whole study group
(22df=0.21,
P=.90). Also, no overall difference in genotype
distribution was seen among cases and controls
(22df=3.71,
P=.16). Logistic regression analysis assuming
additive (AA versus AB versus BB),
dominant (AA and AB versus BB), or
recessive (AA versus AB and BB)
inheritance likewise failed to reveal any significant association
between phenotype and genotype, with ORs (95% CIs) of
1.47 (0.94 to 2.27), 1.41 (0.68 to 2.91), and 1.98 (0.96 to 4.10),
respectively.
Allele frequencies for + and - alleles were 0.52 and
0.48 in case subjects and 0.53 and 0.47 in control subjects,
respectively. Genotype frequencies were 0.252 for ++, 0.525 for
+-, and 0.223 for -- in cases; 0.257 for ++, 0.543 for +-, and
0.200 for -- in controls; and 0.254 for ++, 0.532 for +-, and 0.214
for -- in both groups combined. Genotype frequencies did not
deviate from the Hardy- Weinberg equilibrium in controls
(22df=0.26,
P=.88), cases
(22df=0.19,
P=.91), or the whole study group
(22df=0.43,
P=.81). No overall difference in genotype
distribution was seen among cases and controls
(22df=0.12,
P=.94). Logistic regression analysis assuming
additive (-- versus +- versus ++), dominant (-- and +- versus
++), or recessive (-- versus +- and ++) inheritance likewise failed
to reveal any significant association between phenotype and
genotype, with ORs (95% CIs) of 1.03 (0.65 to 1.62), 0.99
(0.49 to 2.02), and 1.09 (0.51 to 2.34), respectively.
Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
The present study represents the first genetic
investigation of a possible association between genetic variants of the
ACE, MCC, and B2BKR genes and
ACEI-related dry cough. While there is no previous evidence for a
familial aggregation or transgenerational transmission of this
phenotype (understandably so, because of how recently the
phenomenon has been recognized), failure to find any association
between its occurrence and hitherto observed clinical variables
suggests genetically determined susceptibility as one possible
explanation. Our data fail to provide any evidence for the existence of
such an association with the genes/polymorphisms tested.
Selected Abbreviations and Acronyms
ACEI
=
angiotensin-converting enzyme inhibitor
ACE
=
angiotensin-converting enzyme gene
B2BKR
=
B2-bradykinin receptor gene
CI
=
confidence interval
D
=
deletion
I
=
insertion
MCC
=
mast cell chymase gene
OR
=
odds ratio
PCR
=
polymerase chain reaction
RFLP
=
restriction fragment length polymorphism
Acknowledgments
This work was supported by research development award
K04-HL-03138-01 from the National Heart, Lung, and Blood Institute (Dr
Lindpaintner). We would like to express our thanks to Dr Martin G.
Larson for his help with some of the statistical work.
References
Top
Abstract
Introduction
Methods
Results
Discussion
References
1.
SOLVD Investigators. Effect of enalapril on
survival in patients with reduced left ventricular ejection
fraction and congestive heart failure. N Engl J
Med. 1991;325:293–302.[Abstract]
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