From the Human Genetics Center (M.F., E.B.) and the Institute of
Molecular Medicine (E.B.), University of Texas at Houston Health Science
Center; the Department of Epidemiology, University of Texas M.D. Anderson
Cancer Center, Houston (C.I.A.); the Department of Human Genetics, University
of Michigan, Ann Arbor (S.K., C.F.S.); and the Division of Hypertension,
Department of Internal Medicine, Mayo Clinic, Rochester, Minn (S.T.T.).
Correspondence to Eric Boerwinkle, PhD, Human Genetics Center, University of Texas Houston Health Science Center, PO Box 20334, Houston, TX 77225.
Abstract
Background—The
renin-angiotensin system regulates blood pressure through
its effects on vascular tone, renal hemodynamics, and
renal sodium and fluid balance.
Methods and Results—Using data from a large population-based
sample of 1488 siblings having a mean age of 14.8 years and belonging
to the youngest generation of 583 randomly ascertained three-generation
pedigrees from Rochester, Minn, we carried out variance
components–based linkage analyses to evaluate the contribution
of variation in four renin-angiotensin system gene regions
(angiotensinogen, renin, angiotensin
I–converting enzyme, and angiotensin II receptor type 1)
to interindividual variation in systolic,
diastolic, and mean arterial pressure. We
rejected the null hypothesis that allelic variation in the region of
the angiotensin-converting enzyme (ACE) gene
does not contribute to interindividual blood pressure variability.
After conditioning on measured covariates, variation in this region
accounted for 0%, 13% (P=0.04), and 16%
(P=0.04) of the interindividual variance in
systolic, diastolic, and mean arterial
pressures, respectively. These estimates were even greater in a subset
of subjects with a positive family history of hypertension (0%, 29%
[P=0.005], and 32% [P<0.005],
respectively). In sex-specific analyses, genetic variation in
the region of the ACE gene significantly influenced
interindividual blood pressure variation in males (37% for SBP
[P=0.03], 38% for DBP [P=0.04], and
53% for MAP [P<0.005]) but not in females.
Conclusions—Although it is possible that variation in a gene near
the ACE gene may explain the observed results, knowledge
about the physiological involvement of ACE in blood
pressure regulation supports the proposition that the
ACE gene itself influences blood pressure variability in
a sex-specific manner.
Hypertension,
or high BP, is the most common risk factor for myocardial infarction,
stroke, end-stage renal disease, and peripheral vascular
disease.1 2 Despite evidence for a substantial
genetic component underlying interindividual BP variation, progress
toward identifying specific genes has been slow. Genetic linkage
analysis has been the method of choice for identifying genes
contributing to human disease. However, conventional linkage approaches
based on log of the odds score analysis require precise
specification of the genetic model and may, therefore, not be efficient
for the study of a complex trait such as BP. Robust
"nonparametric" linkage approaches, based on allele
sharing, make minimal assumptions regarding the underlying mode of
inheritance and are computationally simple. Thus, they are better
suited for and widely applied to the study of phenotypes having
a multifactorial etiology, such as BP or essential hypertension.
A logical approach to dissecting the genetic determinants of BP is
to examine those genes encoding products with known biological
effects on BP regulation. The RAS is an important system of BP control
through its effects on vascular tone, renal
hemodynamics, and sodium and volume homeostasis
(reviewed in Reference 33 ). Several studies have established that
variation in the genes of the RAS is associated with the pathogenesis
of essential hypertension.4 5 These studies have
primarily focused on severely affected individuals and therefore,
provide incomplete information about the determinants of
interindividual variability in BP in the general population. We
investigated the contribution of genetic variation in the region of
four RAS genes (AGT, REN, ACE, and
AT1) to interindividual variation in BP in a
population-based sample of young whites from Rochester, Minn. Using BP
data from siblings belonging to the youngest generation of 583
pedigrees ascertained without regard to health status from the
population of Rochester, Minn, we tested hypotheses about whether or
not variation within or very near the genes encoding the four major
components of the RAS contributes to interindividual variance in SBP,
DBP, and MAP levels.
Methods
Subjects
For these analyses, SBP and DBP values were the means of three
readings taken at least 2 minutes apart with a random-zero
sphygmomanometer. For each reading, the pressure at the Korotkoff phase
I sound was taken as the SBP. DBP was determined at the Korotkoff phase
IV sound in children <13 years old and at the Korotkoff phase V sound
in all other subjects. MAP was calculated from the averaged SBP and DBP
values as follows: MAP=(SBP+2DBP)/3.
Marker Data Collection
Variance Component Linkage Analyses
The vector of phenotypic values for each sibship was assumed to have a
multivariate normal distribution. A likelihood function
was numerically maximized18 to yield estimates of
the variance components and covariate effects. Robust estimates of the
variance of the maximum-likelihood parameters were computed
as described in Amos et al.14 The
parameter of primary interest is the component of variance,
Results
Descriptive statistics for BP and covariate measures in the sample
of siblings are given by sex in Table 2
For each marker locus, the amount of interindividual variation in the
three measures of BP attributable to genetic variation in each of the
four RAS gene regions, to residual familial effects (including genetic
effects unlinked to the marker locus), and to nongenetic sources of
variation was estimated while jointly accounting for the effects of
measured covariates. The maximum-likelihood estimate of each
parameter and its robust SE are shown in Table 3
Because the impact of genetic variation on interindividual variation in
BP levels may differ among subsets of individuals in the population, we
next examined the subgroup of siblings with a positive family history
of hypertension. Family history of hypertension was defined as those
individuals having at least one parent or grandparent with definite
essential hypertension (ie, requiring antihypertensive treatment or
having an SBP >160 mm Hg or a DBP >95 mm Hg). In this
subset, the contribution of variation in the ACE gene region
to BP variation was even stronger, accounting for 29.1% and 32.7% of
the total variance in DBP and MAP (P=0.005 and
P<0.005), respectively (Table 4
Because there is ample evidence that BP distributions differ between
males and females (eg, see Reference 66 ) and because genetic variation
may have a different effect between the sexes,20
we next estimated the contribution of each of the four marker regions
to interindividual variation in BP levels separately by sex. Results
for the ACE marker are presented in Table 4
Discussion
We have used variance component–based linkage analyses to
estimate the contribution of variation in the regions of the RAS genes
to interindividual variation in BP in a large sample of white sibships
from Rochester, Minn. Sampling from the youngest generation, wherein
the prevalence of hypertension is low, enabled us to examine the
complete distribution of alleles underlying interindividual
variation in the quantitative BP phenotype in the white
population of Rochester, Minn. Such a strategy is not subject to the
limitations associated with the presence of treated essential
hypertension in older generations, which may result in the exclusion of
a large proportion of patients because their measures of BP do not
reflect untreated levels. The power of detecting a genetic effect on BP
variation in a sample lacking these genetically susceptible individuals
may be decreased. Unlike previous studies that focused on restricted
subsets of hypertensive individuals (eg, those with severe or familial
essential hypertension), our strategy was to estimate the impact of
variation in the RAS gene regions in the population at large. Indeed,
identifying a gene with large effects on BP levels in only a small
subset of severely affected individuals does not establish that this
gene will have a significant impact on BP variation in the general
population.
We did not find significant evidence that genetic variation in the
regions of the AGT or REN gene contributes to
interindividual variation of BP in our sample. This finding suggests
that the impact of variation in these genes on interindividual
variability in BP may not be large in the general population of young
white individuals. However, this conclusion does not preclude the
possibility that the contribution of these genes may be greater in some
subset of individuals. Indeed, two studies have demonstrated
significant linkage between the (CA) repeat polymorphism of the
AGT gene and a locus that contributes to severe essential
hypertension in white individuals.4 21 No
evidence of linkage22 or
association23 24 25 has been reported between
variation in the REN gene and BP or essential hypertension
in whites. These results, along with ours, suggest that the
REN gene is unlikely to play a major role in BP variation in
whites. It should be pointed out, however, that the observed
heterozygosity for the REN marker locus was much less than
expected on the basis of the original published
report.10 Although the power of our linkage
analyses was high (>80%) for detecting genes with moderate
effects (contributing 20% of the phenotypic variance), this power was
reduced when the marker locus was less informative, such as the
REN marker locus.
No linkage was demonstrated between a (CA) microsatellite of the
AT1 gene and a locus that influences interindividual BP
variation in our study or a variability in risk of essential
hypertension in a previous study by Bonnardeaux et
al.5 However, a significant association of an
A1166C polymorphism of this gene with severe essential hypertension
was reported in a cross-sectional
analysis.5 Association studies are able
to detect weaker genetic effects, which may be difficult to be
recognized by linkage analysis.26 27
Therefore, we cannot exclude the possibility that the AT1
gene may have a small impact on interindividual variation in BP,
especially among individuals with clinically manifest hypertension.
We found significant evidence to support a contribution of the
ACE gene region to interindividual variation in BP in our
sample of young white individuals. This effect was consistent
across analyses when both the Haseman-Elston (not shown) and
variance component linkage methods were used and was even stronger in a
subset of individuals with a family history of hypertension.
Sex-specific analyses indicated a significant influence of
variation in the ACE gene region on interindividual
variability in all three measures of BP in males but not in females.
For DBP, for example, the ACE gene region accounted for 38%
of the interindividual BP variation in males, whereas this value was
only 5% in females.
ACE plays a key role in the generation of the vasopressor
angiotensin II and in the degradation of the vasodilator
bradykinin. In addition, the efficacy of ACE inhibitors as
therapeutic agents in the treatment of essential hypertension is well
documented.28 Early reports of a strong genetic
linkage between a marker located in the ACE gene region and
salt-loaded DBP in stroke-prone spontaneously hypertensive
rats29 30 first pointed to the ACE
gene as an attractive candidate for the study of human essential
hypertension. An I/D polymorphism located in intron 16 of the gene
is associated with interindividual variance in plasma ACE
activity.31 Conflicting results have been
reported regarding an association between the ACE I/D
polymorphism and either BP or essential
hypertension.32 33 34 35 36 37 However, it is not known at
this time whether the I/D polymorphism is functionally relevant or
whether it is a marker in linkage disequilibrium with a nearby
functional mutation.
The mechanism by which the effect of the ACE gene on BP is
exerted in a sex-specific fashion remains to be more fully
investigated. O'Donnell et al38 have recently
observed a similar significant relationship between ACE gene
region variation and BP in males but not in females. In addition,
O'Donnell et al38 also report that this
sex-specific relationship extends to the clinically defined
hypertension end point. An association between plasma ACE activity and
BP has been demonstrated in males but not in females in a large
population-based study39 and in other groups of
men.40 41 Interestingly, a similar sexual
dimorphism was observed in a mouse model lacking a functional
ACE gene sequence.42 BP was
significantly lower than normal in male but not female mice
heterozygous for the disrupted ACE gene. In addition, Kreutz
et al43 suggested that an interaction between a
putative locus located on the Y chromosome and a locus mapping very
near the ACE gene may partially explain differences in
salt-loaded BP levels observed among spontaneously hypertensive
rats.
A previous report by Jeunemaitre et al8
demonstrated no evidence of linkage between variation at the
ACE locus and a locus contributing to essential hypertension
in a sample of white patients from Utah. Failure to detect an effect of
a particular gene on BP in a moderately sized, clinic-based sample does
not preclude an effect of this gene in other groups of individuals in
the population. The possibility must also be considered that age
modulates the influence of genetic variation in the ACE gene
region on interindividual variation in BP levels. As a result, the
contribution of this region to BP variability may not be the same in
adults and children. The study of Tiret et al,44
which reported a significant association between plasma ACE activity
and BP levels in the offspring but not the parents of 98 healthy,
nuclear families of white origin, is consistent with such a
hypothesis. In preliminary analyses (data not shown) of the
relationship between the ACE I/D polymorphism, plasma
ACE activity, and BP in a subset of the pedigrees analyzed
here, we detected a significant relationship between plasma ACE
activity and BP. Second, there was also a significant relationship
between the ACE I/D polymorphism and SBP, but this
relationship was found to be complex. In particular, we detected
significant interactions between the ACE I/D
polymorphism and body size as they combined to influence BP levels,
and these effects were sex dependent.
Although the ACE gene is the prime candidate for the locus
responsible for the effect on BP variability in our sample, the
present study cannot distinguish between a contribution of the
ACE gene itself and that of other genes closely linked to
the measured polymorphic marker. At least two other genes with
possible influences on BP are located in the region of the
ACE gene on chromosome 17: the
phenylethanolamine-N-methyltransferase and human growth
hormone genes. Phenylethanolamine-N-methyltransferase is an
enzyme involved in the biosynthesis of catecholamines in
the brain.45 46
Phenylethanolamine-N-methyltransferase activity and
epinephrine levels have been found to be significantly elevated
in various hypertensive animal models.47 48 Human
growth hormone has major effects on body size and body composition by
promoting linear growth, increasing lean body mass, and decreasing body
fat.49 The relationship between BP and body size
is well known. Effects of growth hormone associated with increased
sodium retention and plasma volume expansion have also been well
documented.50 51 52 In addition, administration of
low-dose recombinant human growth hormone to growth hormone–deficient
subjects produces many of the features of syndrome X, including
elevated BP.53 54 55 56 Linkage disequilibrium
analysis using DNA sequence variants in and around these
logical candidate genes is being undertaken to confirm the results
presented here and to identify the mutations responsible for
the observed effect.
Selected Abbreviations and Acronyms
Acknowledgments
This work was supported by grants RO1 HL51021, RO1 HL30428, U10
HL54481, U10 HL54457, and U10 HL54464 from the National Heart, Lung and
Blood Institute; grant RO1 GM52607 from the National Institute of
General Medical Sciences; and funds from the Mayo Foundation. The
authors would like to thank Mariza de Andrade, Terry Bertin, Kim
Lawson, Li Li, and Dakai Zhu for their technical assistance.
Footnotes
Guest editor for this article was Suzanne Oparil, MD, University of Alabama at Birmingham.
Received August 27, 1997;
revision received February 5, 1998;
accepted February 10, 1998.
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© 1998 American Heart Association, Inc.
Clinical Investigation and Reports
Variation in the Region of the Angiotensin-Converting Enzyme Gene Influences Interindividual Differences in Blood Pressure Levels in Young White Males
Key Words: linkage analysis • blood pressure • angiotensin-converting enzyme • whites • males
All individuals were members of 583 three-generation pedigrees
taking part in the Rochester Family Heart Study, a population-based
study initiated in 1984 to investigate the role of genetic factors in
the occurrence of hypertension and cardiovascular
disease in Rochester, Minn. Families participating in the study were
ascertained through households having two or more children enrolled in
the schools of Rochester, Minn, and without regard to the health status
of the family members. All individuals provided their informed consent
to participate in the Rochester Family Heart Study. Recruitment and
examination protocols have been described
elsewhere.6 7 From these pedigrees, 1569 full
siblings distributed among 587 sibships and belonging to the youngest
generation were identified. For a large proportion of the sibships
(92%), both parents were available for genotyping. The most common
sibship constellation (47%) was two full siblings, with information on
both parents. Nonwhite individuals and those taking medications that
may have modified BP levels (n=81 individuals) were excluded in the
linkage analyses reported here.
All 3653 members of the 583 Rochester Family Heart Study
pedigrees were genotyped for one polymorphic marker located
within or very near the sequence of each of the genes encoding
angiotensinogen (ie, AGT), renin (ie,
REN), angiotensin I–converting enzyme (ie,
ACE), and the angiotensin II receptor type 1
(ie, AT1) (Table 1
). The
AGT and AT1 markers were dinucleotide
repeat polymorphisms located 12 and 15 kb, respectively, downstream
from the 3' end of the coding gene sequence. The REN marker
was a tetranucleotide repeat polymorphism located in
intron 7 of the REN gene. The ACE marker was a
compound tetranucleotide/dinucleotide repeat
located between the 18th and 19th Alu sequences of the regulatory
region of the human growth hormone gene. This marker showed complete
linkage with the ACE gene in the Centre d'études du
polymorphisme humain (CEPH) families.8 Each
microsatellite repeat polymorphism was amplified by the polymerase
chain reaction using flanking primer pairs and temperature conditions
as previously reported (References 8 through 118 9 10 11 ; Table 1). Size
variation of the amplified products was resolved by
polyacrylamide gel electrophoresis and subsequent
autoradiography. Each genotype was scored
independently by two laboratory personnel, and any discrepancies were
resolved by a third more senior laboratory supervisor.
View this table:
[in a new window]
Table 1. Description of Genetic Markers
Identical by descent (IBD) calculations on extended pedigree
data were carried out using a modified version of the Curtis and
Sham12 algorithm. Allele frequencies at each
marker locus were estimated directly by gene counting from
genotype data obtained in the total sample. Linkage
analyses were carried out using both the regression method of
Haseman and Elston13 and a variance components
approach.14 Similar results were obtained by both
methods. The latter method has been shown to provide greater
statistical power and more precise estimates of the components of
variance attributable to linked and unlinked genetic factors (see
References 14 through 1614 15 16 ). This article, therefore, focuses on results
obtained from maximum-likelihood estimates of the components of
variance as described by Amos et al.14 17 This
variance component–based linkage method was used to estimate the
relative contribution of each of the four RAS loci to interindividual
differences in three measures of BP: SBP, DBP, and MAP. The
quantitative trait was modeled as an additive function of fixed effects
from covariates, random effects from variation at a genetic locus
linked to the marker locus, random familial effects, and residual
sources of variation, including measurement error. The contributions of
genetic loci unlinked to the marker locus and those of nongenetic
factors shared among siblings are confounded in the variance component
referred to as "familial effects." The covariate effects evaluated
were sex (except in sex-specific analyses), age, age squared,
and body mass index. If one assumes no dominance at the linked locus
and no recombination between this locus and the marker locus, the
covariance matrix (
) among individuals in a sibship
h is given by
h=
2a+
2G+I2e,
where is a matrix with elements 
ij
representing the proportion of alleles shared IBD at
the marker locus between individuals i and j in
sibship h; is the matrix of coefficients of
relationship; and I is the identity matrix. 2a, representing
the contribution of the candidate gene region to the phenotypic
variance. The null hypothesis that
2a is equal to zero was
tested by the likelihood-ratio criterion
=-2(L0-L1),
where L1 is the value of the log-likelihood
function evaluated at the maximum-likelihood estimates of the
parameters and L0 is the
maximum of the log-likelihood function evaluated when
2a is constrained to equal
zero. Because estimates of the variance components are constrained to
be nonnegative, the asymptotic distribution of L, under the
null hypothesis, is approximately a 1/2:1/2 mixture of
2 with one degree of freedom and a point mass
at zero.19 In this situation, the critical value
used to assess the significance of the likelihood-ratio test at the
=0.05 level corresponds to the critical value associated with a
significance level of 2 (0.10) for the usual
2 distribution. .
All individuals were white, normotensive (ie, SBP 
140 and DBP 90),
and not taking medication that might have modified BP levels. The mean
ages were 14.55 years for female subjects and 14.96 years for male
subjects and were not significantly different between sexes. Males had
a significantly higher SBP than did females. DBP and MAP levels and
body mass index were not significantly different between the two
sexes.
View this table:
[in a new window]
Table 2. Characteristics of the Sample by Sex . The P value for the
likelihood-ratio test of the significance of genetic variation linked
to the marker locus is also given. After conditioning on covariate
effects, variation in the AGT gene region accounted for
13.0%, 11.1%, and 14.1% of the interindividual variance in SBP, DBP,
and MAP, respectively, but contribution of this region to BP
variability did not reach statistical significance. Similarly, no
evidence for a statistically significant influence of the
REN gene region or the AT1 gene region on
interindividual variation in SBP, DBP, or MAP was obtained in this
large sample of sibships. Point estimates of the contribution of the
REN gene region to interindividual variance in SBP, DBP, and
MAP were 17.5%, 0%, and 2.39%, respectively. These estimates were
11.3%, 10.3%, and 12.7% for the AT1 gene region. A
significant contribution of the ACE gene region to
interindividual variation in both DBP and MAP was detected and
accounted for 13.6% and 16.5% of the variance in DBP and MAP,
respectively. No significant influence of this region on
interindividual variation in SBP was observed.
View this table:
[in a new window]
Table 3. Maximum-Likelihood Estimates±Robust SEs of the
Variance Contribution of the Four RAS Loci to SBP, DBP, and MAP Levels ). No evidence for a contribution of
variation in the ACE gene region to interindividual
variation in SBP was observed. We did not detect a significant effect
of AGT, REN, and AT1 on
interindividual variation in any measure of BP in this subgroup of
individuals with a family history of hypertension (not shown).
View this table:
[in a new window]
Table 4. Maximum-Likelihood Estimates±Robust SEs of the
Variance Contribution of the ACE Locus to SBP, DBP, and MAP
Levels in Subsets of Siblings 
. A
significant contribution of the ACE gene region was
present in males but not in females for all three measures of BP.
These analyses indicated that 37.5% of the variance in SBP
(P=0.03), 38.4% of the variance in DBP (P=0.04),
and 53.5% of the variance in MAP (P<0.005) was
attributable to variation in or around the ACE gene in male
siblings only. No evidence for an effect of the AGT,
REN, and AT1 gene regions on interindividual
variation in SBP, DBP, or MAP was detected in either males or females
(data not shown).
ACE
=
angiotensin-converting enzyme (gene)
AGT
=
angiotensinogen (gene)
AT1
=
angiotensin II type 1 receptor (gene)
DBP
=
diastolic blood pressure
I/D
=
insertion/deletion
MAP
=
mean arterial pressure
RAS
=
renin-angiotensin system
REN
=
renin (gene)
SBP
=
systolic blood pressure
View this table:
[in a new window]
Table 41. Maximum-Likelihood Estimates±Robust SEs of the
Variance Contribution of the ACE Locus to SBP, DBP, and MAP
Levels in Subsets of Siblings
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