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Genome-Wide Scan for Pulse Pressure in the National
Heart, Lung and Blood Institute’s Framingham Heart Study
Anita L. DeStefano, Martin G. Larson, Gary F. Mitchell, Emelia J. Benjamin, Ramachandran S. Vasan,
Jiang Li, Diane Corey, Daniel Levy
Downloaded from http://hyper.ahajournals.org/ by guest on June 18, 2017
Abstract—The objective of this study was to assess heritability and identify chromosomal regions showing evidence of
linkage to pulse pressure (PP), a simple indicator of proximal conduit vessel stiffness. Blood pressure data were analyzed
for 8478 members of the National Heart, Lung and Blood Institute’s (NHLBI) Framingham Heart Study. Long-term PP
was defined using 2-stage analysis. First, the difference between systolic and diastolic blood pressure was averaged over
all qualifying clinic examinations. In the second stage, mean PP was adjusted for mean age, time period of examination,
and body mass index by regression analysis. PP values were available for 6421 individuals in 1593 families for
heritability estimation and for 2492 individuals in 330 families for linkage analysis. Microsatellite markers covering the
genome at 10 cM intervals were typed by the NHLBI Mammalian Genotyping Service; genome scan data were available
on 1585 individuals with PP data. Heritability estimates of long-term PP accounting for hypertension treatment and for
ignoring treatment were 0.52 and 0.51, respectively. Variance component linkage analysis identified several locations
with suggestive evidence of linkage: chromosome 15 at 122 cM (logarithm of odds [LOD]⫽2.94), chromosome 7 at 71
cM (LOD⫽2.42), chromosome 5 at 53 cM (LOD⫽2.03), and chromosome 10 at 81 cM (LOD⫽1.83) for PP accounting
for treatment. LOD scores were slightly lower when ignoring treatment, with the exception of a peak on chromosome
10 at 81 cM (LOD⫽2.58). In conclusion, we have demonstrated a substantial genetic component to PP and have
identified 4 chromosomal regions that may harbor genes influencing vascular stiffness. (Hypertension. 2004;
44:152-155.)
Key Words: genetics 䡲 blood pressure 䡲 human
P
Subjects
ulse pressure (PP), which is the difference between
systolic blood pressure (SBP) and diastolic blood pressure (DBP), provides an indirect measure of pulsatile hemodynamic load and vascular stiffness. PP is associated with left
ventricular hypertrophy,1 carotid intimal-medial thickness,2,3
and artherosclerotic burden.4 Of the 3 BP components, PP has
also been found to be the best predictor of risk for cardiovascular disease5 and heart failure in older people.6 An
understanding of the genetic determinants of PP, therefore, is
important.
Prior genetic studies in Nigerian,7 Mexican American,8 and
Utah cohorts9 have found modest heritability for PP, ranging
from 0.12 to 0.25. In these 3 studies, PP was measured at a
single point in time. In this study, we estimated heritability of
PP and performed a genome-wide linkage analysis based on
repeated measures obtained during up to 50 years of
follow-up of Framingham Heart Study participants.
Subjects were participants in the Framingham Heart Study, a
longitudinal study established in 1948 with the enrollment of 5209
individuals residing in Framingham, Mass.10 Beginning in 1971,
5124 offspring of the original participants and their spouses were
recruited.11 At each clinic visit, the physician measured the seated
BP with a mercury column sphygmomanometer. The systolic and
diastolic pressures were determined by the first and fifth Korotkoff
sounds, respectively. Two BP measurements were averaged to derive
the systolic and diastolic pressures for that examination, except for
the first offspring examination at which only 1 BP measurement was
obtained. All subjects gave informed consent, and the study protocol
was approved by the Institutional Review Board at Boston Medical
Center (Mass).
PP Phenotype
Long-term PP was defined as the mean difference between SBP and
DBP obtained at each qualifying examination. The method used to
compute long-term blood pressure and to impute SBP and DBP in
the setting of antihypertensive treatment has been described previously.12 Long-term BP data were analyzed for 8478 subjects who
met criteria using a 2-stage procedure: (1) within-subject mean SBP
and DBP were calculated and (2) sample-wide regressions were used
to adjust mean PP (defined as mean SBP⫺mean DBP) for age, time
Methods
An expanded Methods section is available in the online supplement
at http://www.hypertensionaha.org.
Received March 17, 2004; first decision April 5, 2004; revision accepted May 28, 2004.
From the Department of Biostatistics (A.L.D., J.L.), Boston University School of Public Health, Mass; the Departments of Neurology (A.L.D.) and
Epidemiology and Preventive Medicine (M.G.L., E.J.B., R.S.V., D.L.), Cardiology Section (E.J.B., R.S.V., D.L.), Boston University School of Medicine,
Mass; the National Heart, Lung, and Blood Institute’s Framingham Heart Study (M.G.L., E.J.B., R.S.V., D.C., D.L.), Framingham, Mass; and
Cardiovascular Engineering, Inc. (G.F.M.), Holliston, Mass.
Correspondence to Anita L. DeStefano, Boston University School of Public Health, 715 Albany Street, T418E, Boston, MA 02118. E-mail
[email protected]
© 2004 American Heart Association, Inc.
Hypertension is available at http://www.hypertensionaha.org
DOI: 10.1161/01.HYP.0000135248.62303.81
152
DeStefano et al
period of examination, and body mass index, yielding a residual for
each subject. Residuals were computed separately for each sex and
cohort.
For subjects receiving hypertension treatment, the recorded BP is
less than what the untreated value would be. We addressed treated
observations by using a nonparametric algorithm to adjust BPs for
treatment effect as previously described.12 These adjusted BP values
were used in place of the measured BP values when obtaining the
difference between SBP and DBP. Analyses were also conducted on
long-term average PP ignoring treatment.
Genome-Wide Scan for Pulse Pressure
153
Variance Component Multipoint LOD Scores >1.5 for
Long-Term Pulse Pressure Obtained From Genehunter
Adjusted for
Treatment
Chromosome
cM
Ignoring Treatment
LOD
cM
LOD
1.51
5
53
2.03
56
7
71
2.42
70
2.36
7
152
1.54
154
1.55
Genotyping
8
140
1.56
140
1.22
DNA was extracted using a standard protocol. A 10 cM density
genome scan was performed (marker set 8A, average heterozygosity
0.77) on 1702 individuals with DNA available from the 330 largest
families by the National Heart, Lung and Blood Institute Mammalian
Genotyping Service laboratory at the Marshfield Clinic (Marshfield,
Wis). Genotype data were cleaned as previously described.12
10
81
1.83
80
2.58
15
122
2.94
122
2.86
22
36
1.75
36
1.02
Results
by-descent estimation at this location may not be as accurate
as if there were additional flanking markers typed. However,
such genotype data are currently not available in these
subjects.
LOD scores ⬎2.0 for PP accounting for treatment were
also observed on chromosome 5 at 53 cM (LOD⫽2.03) and
chromosome 7 at 71 cM (LOD⫽2.42). Minor peaks (LOD
between 1.5 and 2.0) were observed on chromosomes 7, 8,
10, and 22. LOD scores tended to decrease slightly when
treatment was ignored. An exception to this pattern was the
chromosome 10 linkage peak at 80 cM, which yielded a LOD
score of 2.58 when treatment was ignored.
A permutation approach that maintains the trait heritability17 was implemented to assess the significance of the largest
observed LOD score (2.94). The heritability of the permuted
phenotypes averaged over 1000 replicates was 0.51, indicating that, on average, the permutated data sets maintained the
additive genetic variance. The genome-wide empirical P
value associated with LOD⫽2.94 is 0.067.
For heritability estimation, long-term average PP was available for 6421 subjects in 1593 families. Linkage analysis was
based on the long-term average PP values of 2492 subjects
from the 330 families in which the genome scan was
performed. There were 1584 individuals with genotype and
PP data available.
Among the 1584 individuals with both genotype information and phenotypic measures, mean PP was
47.33⫾10.77 mm Hg when accounting for treatment effect
and 47.09⫾9.56 mm Hg when ignoring treatment. Descriptive statistics for other characteristics in these subjects have
been previously published.12 Estimated heritability for mean
PP accounting for treatment effect was 0.52⫾0.03. Heritability ignoring antihypertensive treatment was nearly identical at
0.51⫾0.03. Thus, we have demonstrated a considerable
genetic influence for PP.
Genome scan results are summarized in the Table and
Figure. The highest multipoint LOD score for PP accounting
for treatment was 2.94 observed on chromosome 15 (122
cM). This value is just below the threshold for significant
linkage on a genome-wide level and must be considered
strong suggestive evidence of linkage to this chromosome.
There was minimal change in the LOD score at this location
when treatment was ignored (LOD⫽2.86). This peak does
occur for the last marker on the chromosome, and identity-
We found highly suggestive evidence of linkage for PP on
chromosome 15 (LOD score 2.94 at 122 cM). This is the
same region where linkage to low DBP (LOD⫽3.77) was
previously reported in a Chinese population.18 This overlap is
particularly intriguing because a low diastolic pressure is
often a key component of an elevated PP.
A genome-wide linkage analysis of PP in a populationbased sample of Mexican Americans (San Antonio Family
Heart Study) revealed suggestive evidence of linkage (LODs
between 2.78 and 1.95) for 4 markers: D7S1799 (113.9 cM),
D8S1100 (154 cm), D18S844 (116.4 cM), and D21S1440
(36.7 cM).8 The chromosome 8 location identified in that
study (LOD⫽0.59 at D8S1179 at 135 cM and LOD⫽1.98 at
D8S1100 at 154 cM) overlaps with the region on chromosome 8 that yielded a LOD score of 1.56 (at 140 cM) in our
Framingham sample. As noted by Atwood et al,8 this region
on chromosome 8 contains the aldosterone synthase gene
(CYP11B2) and the adjacent 11 ␤-hydroxylase gene, which
are causative for glucocorticoid-remediable aldosteronism, a
rare Mendelian form of hypertension.19
Linkage analysis of PP in extended Utah pedigrees ascertained on the basis of early coronary disease deaths or strokes
or early-onset hypertension yielded suggestive evidence of
Statistical Analysis
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Heritability was estimated by a variance component model using the
program SOLAR.13 Multipoint linkage analysis was conducted using
the variance component approach as implemented in the program
Genehunter.14,15 Although 14 families were split because of computational constraints when using Genehunter, the algorithm implemented in Genehunter has been shown to provide more exact
multipoint identity-by-descent estimates.16 The splitting of families
resulted in linkage analysis being performed on 345 pedigrees
compared with the 330 originally defined.
Variance components methods have been shown to be sensitive to
departures from normality. Although extreme departures from normality were not observed for these data (skewness⫽0.76, kurtosis⫽1.70 for residual PP adjusted for treatment), a constrained
permutation test, which maintains the additive genetic variance of
the phenotype,17 was used to obtain a genome-wide empirical P
value for the largest logarithm of odds (LOD) score observed.
Simulation, permutation, and linkage analysis was repeated 1000
times.
Discussion
154
Hypertension
August 2004
Genome-wide linkage analysis results for long-term
pulse pressure. Multipoint
LOD score is on the vertical
axis, and centiMorgan distance from the p-terminus of
the chromosome is on the
horizontal axis.
Downloaded from http://hyper.ahajournals.org/ by guest on June 18, 2017
linkage (LOD ⬎1.5) for 11 chromosomal regions. A LOD
score of 1.53 was observed at 8q at 164 cM.9 This linkage
peak overlaps with the 8q peak observed in Mexican Americans and is approximately 24 cM from the chromosome 8
linkage peak observed in the Framingham Heart Study
sample. The Framingham Heart Study and the Utah pedigrees
also had concordance for linkage on chromosome 7. The Utah
study observed linkage at 70 cM (LOD⫽1.59), which overlaps with the peak at 71 cM (LOD⫽2.42) in the current study.
No other linkage peaks were common between the Framingham Heart Study and either the San Antonio Family
Heart Study or the Utah study. This is not surprising given the
ethnic differences between the populations and the differences in study design (cross sectional for San Antonio and
Utah and longitudinal for Framingham). Additionally, distinct linkage analysis methods were applied in each study: a
multipoint variance component approach in the current genome scan, a 2-point parametric model in the San Antonio
analysis, and a multipoint parametric model accounting for
heterogeneity in the Utah sample. The parametric model
applied in the San Antonio study assumed a genetic model
with sex-specific genotypic means and sex-specific and
genotype-specific means for age, age squared, and BMI. This
type of modeling is computationally intensive for large
pedigrees, and computational restraints limited the San Antonio study to 2-point analysis (ie, examining linkage between PP and a single genetic marker at a time).8 The
variance component method used in our study enabled
multipoint analysis in which information from all markers
along a chromosome are used simultaneously to evaluate
linkage. Analysis of the Utah pedigrees was also based on
multipoint inheritance probabilities and required estimation
of additional parameters to specify the trait distribution for
the parametric analysis.9
Linkage analysis to SBP and DBP has been previously
reported in the Framingham Heart Study.12 Minimal overlap
was observed in linkage peaks for SBP (or DBP) and PP. The
peak on chromosome 5 observed for PP (LOD⫽2.03) coincides with a 2-point LOD score of 2.0 we previously observed
for SBP. The peak regions for SBP (LOD⫽4.70 at 67 cM on
chromosome 17) and DBP (LOD⫽2.1 at 7 cM on chromosome 18) yielded LOD scores of 1.47 and 0.0, respectively
for PP. This suggests that genes contributing to variation in
PP, and by inference, to vascular stiffness, may be distinct
from those contributing specifically to SBP or DBP.
Many genes located in the regions for which linkage peaks
were observed can be postulated as candidate genes. We
highlight the most promising in the 4 regions that yielded
suggestive evidence of linkage. Located at the distal end of
the long arm of chromosome 15 are genes for the insulin-like
growth factor-1 receptor (IGF1R); myocyte-specific enhancer
factor 2A (MEF2A), a transcription factor involved in angiotensin II-mediated activation of vascular smooth muscle
cells20; chondroitin synthase (CHSY1), an enzyme involved
in synthesis of proteoglycans that are an integral component
of the extracellular matrix21; and a secretory proprotein
convertase (PACE4).
The genes for 2 major binding proteins of IGF1, IGFBP1
and IGFBP3, are both located in the proximal linkage peak on
chromosome 7. Growth hormone (GH), acting at the GH
receptor located under the chromosome 5 linkage peak,
increases levels of IGF1. Clearly, the GH/IGF axis has
DeStefano et al
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important effects on vascular growth and tone, and several
elements of this axis (eg, IGFR, IGFBP1, IGFBP3, GHR)
have shown up under our linkage peaks, making detailed
analysis of the members of this pathway a high priority for
future studies. There were no obvious candidate genes in our
linkage peak on chromosome 10.
A strategy for moving to gene identification from linkage
analysis results is using a bioinformatics approach to identify
likely candidate genes, such as those described above in the
linkage regions. Selection of candidate genes may be followed by SNP typing within the candidate genes, including
the 3⬘ untranslated region that may be critical for gene
regulation. Family-based association analysis, using the linkage subjects, or association analysis in a subset of unrelated
individuals can then be used to determine whether variants in
the candidate genes influence PP. Given that linkage peaks
tend to be broad and there are numerous genes, which may
have unknown function, it may be necessary to type additional genes or densely cover the entire region with SNP
genotyping. Definitive proof of any positive association
would require functional studies of the specific variant
implicated as well as replication in another population.
Despite the large sample size, extended pedigrees, and detailed phenotyping in the Framingham Heart Study, the largest
LOD score observed in this analysis was 2.94, which highlights
the challenges of genetic localization and identification for
complex traits. Researchers must question whether the best use
of limited resources is to allocate them for follow-up of a modest
linkage peak by either examining candidate genes within the
region or undertaking large-scale sequencing or typing of single
nucleotide polymorphisms across the region for association
studies. Although differences in populations, ascertainment, and
analytic methods make comparison among multiple linkage
analyses difficult, regions such as 8q, which yield suggestive
peaks in multiple studies, may be important regions worthy of
such follow-up. These results also suggest that even larger
family-based studies may be needed.
Perspectives
In summary, we have demonstrated a substantial genetic component for PP. Although we did not meet the Lander and
Kruglyak criterion for genome-wide significance (LOD score
⬎3.2),22 we did observe several suggestive regions of linkage,
most notably LOD⫽2.94 at 122 cM on chromosome 15. A
region on 8q also appears to be of particular interest for PP
because this location yielded suggestive evidence of linkage in 3
independent studies. There are several interesting candidate
genes in the regions of suggestive linkage, and future work will
include association studies of these genes. The third generation
of Framingham Heart Study families are currently being recruited, and extension of these pedigrees by an additional
generation will result in a substantial increase in power for
detecting genes influencing a complex trait such as PP.
Acknowledgments
The Framingham Heart Study is funded by National Institutes of
Health (NIH) contract NO1-HC-25195. This study was also supported by the Donald W. Reynolds Foundation. We thank Ming-Huei
Chen for computing assistance in obtaining the empirical
probability values.
Genome-Wide Scan for Pulse Pressure
155
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Genome-Wide Scan for Pulse Pressure in the National Heart, Lung and Blood Institute's
Framingham Heart Study
Anita L. DeStefano, Martin G. Larson, Gary F. Mitchell, Emelia J. Benjamin, Ramachandran S.
Vasan, Jiang Li, Diane Corey and Daniel Levy
Downloaded from http://hyper.ahajournals.org/ by guest on June 18, 2017
Hypertension. 2004;44:152-155; originally published online June 21, 2004;
doi: 10.1161/01.HYP.0000135248.62303.81
Hypertension is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2004 American Heart Association, Inc. All rights reserved.
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