Genetics
Tyrosine Hydroxylase, the Rate-Limiting Enzyme in
Catecholamine Biosynthesis
Discovery of Common Human Genetic Variants Governing Transcription,
Autonomic Activity, and Blood Pressure In Vivo
Fangwen Rao, MM*; Lian Zhang, MD*; Jennifer Wessel, PhD; Kuixing Zhang, MD, PhD;
Gen Wen, MD, PhD; Brian P. Kennedy, PhD; Brinda K. Rana, PhD; Madhusudan Das, PhD;
Juan L. Rodriguez-Flores, MS; Douglas W. Smith, PhD; Peter E. Cadman, MD; Rany M. Salem, MPH;
Sushil K. Mahata, PhD; Nicholas J. Schork, PhD; Laurent Taupenot, PhD; Michael G. Ziegler, MD;
Daniel T. O’Connor, MD
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Background—Tyrosine hydroxylase (TH) is the rate-limiting enzyme in catecholamine biosynthesis. Does common
genetic variation at human TH alter autonomic activity and predispose to cardiovascular disease? We undertook
systematic polymorphism discovery at the TH locus and then tested variants for contributions to sympathetic function
and blood pressure.
Methods and Results—We resequenced 80 ethnically diverse individuals across the TH locus. One hundred seventy-two
twin pairs were evaluated for sympathetic traits, including catecholamine production, reflex control of the circulation,
and environmental (cold) stress responses. To evaluate hypertension, we genotyped subjects selected from the most
extreme diastolic blood pressure percentiles in the population. Human TH promoter haplotype/reporter plasmids were
transfected into chromaffin cells. Forty-nine single-nucleotide polymorphisms were discovered, but coding region
polymorphism did not account for common phenotypic variation. A block of linkage disequilibrium spanned 4 common
variants in the proximal promoter. Catecholamine secretory traits were significantly heritable (h2), as were stressinduced blood pressure changes. In the TH promoter, significant associations were found for urinary catecholamine
excretion and for blood pressure response to stress. TH promoter haplotype 2 (TGGG) showed pleiotropy, increasing
both norepinephrine excretion and blood pressure during stress. Coalescent simulations suggest that TH haplotype 2
likely arose ⬇380 000 years ago. In hypertension, 2 independent case-control studies (1266 subjects with 53% women
and 927 subjects with 24% women) replicated the effect of C-824T in the determination of blood pressure.
Conclusions—We conclude that human catecholamine secretory traits are heritable, displaying joint genetic determination
(pleiotropy) with autonomic activity and finally with blood pressure in the population. Catecholamine secretion is
influenced by genetic variation in the adrenergic pathway encoding catecholamine synthesis, especially at the classically
rate-limiting step, TH. The results suggest novel pathophysiological links between a key adrenergic locus, catecholamine
metabolism, and blood pressure and suggest new strategies to approach the mechanism, diagnosis, and treatment of
systemic hypertension. (Circulation. 2007;116:&NA;-.)
Key Words: catecholamines 䡲 genetics 䡲 nervous system, autonomic 䡲 nervous system, sympathetic 䡲 norepinephrine
T
yrosine hydroxylase (TH) is the rate-limiting enzyme in
catecholamine biosynthesis.1,2 Substantial loss of TH
enzymatic activity as a consequence of rare inactivating
mutations has profound consequences in humans1 and in mice
with targeted ablation of the TH locus.3
Editorial p ●●●
Clinical Perspective p ●●●
The human TH locus also displays more common natural
allelic variation such as the tetranucleotide repeat [or micro-
Received January 18, 2007; accepted May 8, 2007.
From the Departments of Medicine (F.R., L.Z., K.Z., G.W., B.P.K., M.D., J.L.R.-F., P.E.C., R.M.S., S.K.M., L.T., M.G.Z., D.T.O.), Pharmacology
(D.T.O.), Psychiatry (J.W., B.K.R., N.J.R.), and Biology (D.W.S.) and the Center for Human Genetics and Genomics (N.J.S., D.T.O.), University of
California at San Diego, and the VA San Diego Healthcare System (S.K.M., D.T.O.), San Diego, Calif.
*The first 2 authors contributed equally to the research.
The online Data Supplement, which includes Methods, tables, and figures, can be found with this article at http://circ.ahajournals.org/
cgi/content/full/CIRCULATIONAHA.106.682302/DC1.
Correspondence to Daniel T. O’Connor, MD, Michael G. Ziegler, MD, or Laurent Taupenot, PhD, Department of Medicine and CHGG, UCSD School
of Medicine, 9500 Gilman Dr, La Jolla, CA 92093– 0838. E-mail [email protected], [email protected], or [email protected]
© 2007 American Heart Association, Inc.
Circulation is available at http://circ.ahajournals.org
DOI: 10.1161/CIRCULATIONAHA.106.682302
1
2
Circulation
August 28, 2007
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satellite polymorphism (TCAT)n] in its first intron,4 which
has been used to probe the role of TH in psychiatric
illnesses.5–7 This microsatellite also may be associated with
essential hypertension.4 Previously, we found that the common hepta-allelic (TCAT)n polymorphism predicted alterations in human autonomic function.8 However, is this
intronic variant in itself functional? In transfections,9 the
(TCAT)n repeat silences transcription in a copy number–
dependent way; in contrast, we observed in vivo directionally
opposite associations of common (TCAT)n alleles with autonomic function: (TCAT)10i with activation and (TCAT)6 with
diminution of sympathetic outflow. Therefore, we sought
additional causative/functional variation at the TH locus.
The spectrum of allelic variation at human TH is currently
unknown; therefore, we undertook systematic polymorphism
discovery at the locus. To probe the impact of TH variation on
stress-induced disease pathways, we resequenced ⬇1.2 kbp
of the 5⬘ promoter and all 13 exons and adjacent intronic
regions in 80 ethnically diverse subjects and 422 twins. Twin
pairs enabled us to study whether TH allelic variation
contributed to heritable control of the circulation.
Our results suggest that common variation in the TH
proximal promoter contributes to heritable alteration in multiple autonomic traits, biochemical and physiological, and the
ultimate disease trait of hypertension.
Methods
The following categories are described extensively in the Methods
section of the online Data Supplement: subjects and clinical characterization, genomics, biochemical phenotyping in twin pairs (catecholamines), physiological/autonomic phenotyping in twin pairs in
vivo, and TH promoter haplotype activity in vitro. Further details on
subjects and clinical characterization also are given in the online
Methods section.
Subjects and Clinical Characterization
Polymorphism Discovery
Subjects (n⫽80) resequenced across the TH locus are described in
the online Data Supplement. Their biogeographic ancestries were as
follows: 23 European (white), 25 sub-Saharan African (black), 16
east Asian, and 16 Mexican American (Hispanic).
Twin Pairs
The 172 twin pairs (119 monozygotic, 53 dizygotic; age, 15 to 84
years) are described extensively in the Data Supplement.
Hypertension and Population Blood Pressure Extremes
Subjects with hypertension, both the initial and replication samples,
are described in the online Data Supplement. In the initial sample,
1266 subjects with the highest (4.9th percentile) and lowest (4.8th
percentile) diastolic blood pressures (DBPs) in the population were
evaluated; 53% were female. In the follow-up study, 927 subjects
(essential hypertension versus normal BP) were evaluated; 24% were
female.
Statistical Genetic Analyses
Descriptive statistics (mean, SE) were computed across all of the
twins with generalized estimating equations (PROC GENMOD) in
SAS (SAS, Cary, NC) to account for correlated trait values within
each twinship using an exchangeable correlation matrix.10
Heritability of Phenotype Expression in Twin Pairs
In Vivo
Heritability (h2) is the fraction of phenotypic variance accounted for
by genetic variance (h2⫽VG/VP). Estimates of h2 were obtained by
using the variance component method implemented in the Sequential
Oligogenic Linkage Analysis Routines (SOLAR) package.11 This
method maximizes the likelihood assuming a multivariate normal
distribution of phenotypes in twin pairs (monozygotic versus dizygotic), with a mean dependent on a particular set of explanatory
covariates. The null hypothesis (H0) of no heritability (h2⫽0) is
tested by comparing the full model, which assumes genetic variation,
and a reduced model, which assumes no genetic variation, using a
likelihood ratio test. Covariates (sex and age) significant at P⬍0.05
were retained in the heritability models.
Haplotypes and Linkage Disequilibrium
Haplotypes were inferred from unphased diploid genotypes with the
software package PHASE,12 assigning the 2 most likely haplotypes
to each diploid individual. We inferred the TH promoter haplotypes
using 10 single-nucleotide polymorphisms (SNPs) discovered by
resequencing 293 unrelated individuals (n⫽586 chromosomes) chosen to span 4 diverse ethnic groups: white (European ancestry), black
(sub-Saharan African ancestry), Hispanic (Mexican American), and
east Asian. Blocks of pairwise (SNP-by-SNP) linkage disequilibrium
(LD) were displayed using graphical overview of LD.13
Association
Association studies were performed for the common TH promoter
alleles (minor allele frequency ⬎5%). Each study subject was
categorized according to diploid genotype at a biallelic SNP locus or
carrier status (2, 1, or 0 copies) of a particular TH SNP, haplotype,
or diploid haplotype (diplotype). Unpaired t tests evaluated the
significance of the in vitro haplotype-specific TH promoter activity.
Pleiotropy: Bivariate Genetic Analyses
Pleiotropy (genetic covariance for 2 correlated, heritable traits14) was
estimated as the parameter ␳G in SOLAR.15 As a test of pleiotropy,
bivariate analyses in SOLAR11 (www.sfbr.org/solar, chapter 9.2)
were done to test whether genotype coordinately influenced 2
dependent variables (traits), biochemical (eg, catecholamines) and
hemodynamic (eg, stress BP responses), using nested log-likelihood
values for the bivariate model in the presence or absence of the
genotype: ⫺2(⌬log likelihood)⫽␹2 at df ⫽1. Hardy-Weinberg equilibrium was assessed with a ␹2 goodness-of-fit test using 1 individual
from each twin pair.
Coalescent and Phylogeny Reconstruction
An approximation of the likely age of an SNP mutation (within a
haplotype) in the human lineage was generated by constructing a
coalescent tree16 for SNPs in genetic regions that showed a significant association between SNP haplotype and autonomic function
with the coalescent software package GENETREE.17 GENETREE
permits the construction of coalescent trees inferring the time to most
recent common ancestors of sets of SNP haplotypes. The “root” of
the coalescent tree (the ancestral alleles/haplotype) was specified by
the chimpanzee variant17 because the chimpanzee is the contemporary nonhuman primate with the closest evolutionary ties to humans
(divergent lineages, ⬇4 to 6 million years ago. The resulting
coalescent units were transformed into estimates of years in the past
(time to most recent common ancestors) through adjustment for
expected single base mutation rate per generation, effective human
population size, generation length, and ploidy.16 The approximate
time at which each haplotype arose (from its most recent common
ancestors) was calculated from this equation and assumptions: age
(years)⫽(coalescent units from GENETREE)⫻(20 years/
generation)⫻[2(diploid genome)]⫻(effective population size of
10 000).
Multiple Comparisons
To adjust for the possibility of multiple comparisons when testing the
effect of 4 TH promoter SNPs on autonomic traits, we used the
method of SNP spectral decomposition (SNPSpD) proposed by
Nyholt18 and implemented at http://genepi.qimr.edu.au/general/
daleN/SNPSpD/ to yield an “effective” number of markers within a
block of LD. For this purpose, we used TH promoter SNP data from
1 member of each twinship (ie, 1 founder per family). This method
Rao et al
takes into account intermarker correlations in calculating a new
experiment-wide threshold to keep the type I error rate at ⱕ5%, for
a single phenotype.
Permutation Tests
To enable model-free analyses without reliance on standard asymptotic assumptions, we also used more computationally intensive
permutation tests through the use of the recursive algorithm of Mehta
and Patel19 as implemented by Clarkson et al.20 Trait values were
dichotomized (or converted into 2 quantiles) to allow construction of
3⫻2 contingency tables (diploid genotype by trait); the test is
implemented online at http://www.physics.csbsju.edu/stats/exact.
html.
The authors had full access to and take full responsibility for the
integrity of the data. All authors have read and agree to the
manuscript as written.
Results
TH Genomics: Systematic Human
Polymorphism Discovery
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To identify genetic variants in TH, we resequenced all 13
exons and adjacent intronic regions and 1.2 kbp of 5⬘
promoter (online Table I) from 80 ethnically diverse subjects.
Later, we resequenced the promoter from an additional 213
twin pairs.
Online Figure I illustrates sequence tracings for 4 common
(minor allele frequency ⬎10%) SNPs discovered in the TH
proximal promoter: C-824T, G-801C, A-581G, and G-494A.
Figure 1A shows the local genomic region resequenced.
Forty-nine SNPs and 1 tetranucleotide repeat were identified.
Ten SNPs were located in the ⬇1-kbp proximal promoter, 14
in coding regions, and 25 in untranslated regions or exonadjacent intronic regions. Of the 49 SNPs, 13 were common
(minor allele frequency ⬎5%), including 4 SNPs in the
proximal promoter, 2 in coding regions, 4 in untranslated
regions, and 4 in introns. Among 14 coding region polymorphisms, 11 specify amino acid substitutions. Val81Met is
the most common, with a minor allele frequency at 37.4%;
synonymous Lys240Lys is the second most common at
23.6%. Unusual/rare coding SNPs (all ⬍1.2%, most at 0.3%)
were the balance: Ala6Thr, Arg15His, Ala17Val, Ala23Thr,
Arg89Arg, Ala143Thr, Thr225Asn, Arg410Trp, Asp467Asp,
Val468Met, Ala492Val, and Gly497Asp.
LD Across the TH Locus
Pairwise LD between each common SNP across TH was
quantified as D’, scaled from 0 to 1. Visual inspection (Figure
1B) of plots of the LD structure across TH reveals 2 blocks of
particularly high LD (D’ ⬎0.9): at the 5⬘ (promoter) and mid
to 3⬘ (intron A3exon 12) regions of the gene.
TH Promoter Variants
Ten biallelic variants were discovered (online Table I and
Figure 1A; major allele/position upstream of the ATG/minor
allele): C-833T, C-824T, G-814A, G-801C, T-741C,
A-641G, A-581G, G-494A, C-388T, and G-94T. Alleles at
each SNP were in Hardy-Weinberg equilibrium (determined
within the largest ethnic group, white). Four of 10 SNPs
(C-824T, G-801C, A-581G, and G-494A; shown in online
Figure I) had relatively common minor alleles (⬎10% frequency); these 4 occurred within a span of only 331 bp in the
proximal promoter. Five of 6 uncommon (⬍5%) promoter SNP
Functional Variants of TH
3
alleles were each found in only 1 ethnic group (C-833T,
G-814A, T-741C, C-388T, and G-94T; online Table I). Even the
more common SNP genotype frequencies differed by ethnicity,
with the greatest differences between Asians and blacks at
C-824T (16.7% versus 60.3%), A-581G (10% versus 59%), and
G-494A (71.7% versus 17.9%). Eight of 10 promoter SNPs (all
but G-801C and G-94T) were either purine/purine (A/G) or
pyrimidine/pyrimidine (C/T) transitions.
Twin Phenotyping: Descriptive Statistics
and Heritability
Online Table II describes the twin subject population (n⫽344
individuals). Female subjects (n⫽267) had lower basal SBP
(P⫽0.0438), plasma epinephrine (P⫽0.0065), urinary epinephrine (P⫽0.0284), and norepinephrine (P⫽0.0192) values
than male subjects (n⫽77), consistent with previous reports.21
Older subjects (age ⱖ40 years, n⫽180) had higher basal
SBP (P⬍0.0001) and DBP (P⬍0.0001) and poststress SBP
(P⬍0.0001) and DBP (P⫽0.0008) values than younger
subjects (n⫽164). Older subjects had lower baroreceptor
slope, during both upward (P⬍0.0001) and downward
(P⬍0.0001) deflections. Plasma norepinephrine was increased (P⫽0.0045) in older subjects.
Online Table III presents correlations between variables. In
general, similar correlations were obtained with the parametric and nonparametric methods. Because of the effects of sex
and age (online Table II), further inferential statistics were
performed on age- and sex-adjusted data. Urinary norepinephrine excretion correlated directly with basal SBP, DBP,
and poststress SBP and inversely with baroreceptor slopes,
both downward (␳⫽⫺0.278, P⬍0.001) and upward deflections (␳⫽⫺0.303, P⬍0.001).
Heritability (h2; see Methods) estimates from twin pairs are
shown in online Table IV. Both plasma catecholamine and
urinary catecholamines were significantly heritable, with the
most prominent values for plasma norepinephrine
(h2⫽69.9⫾4.4%; P⬍0.0001) and urinary epinephrine excretion (h2⫽66.7⫾5.9%; P⬍0.0001).
Basal BP and heart rate displayed significant heritability,
with heart rate substantially more heritable (at h2⫽61⫾6%;
P⬍0.0001) than either systolic BP (SBP; h2⫽26⫾8%;
P⫽0.0016) or DBP (h2⫽18⫾9%; P⫽0.0359). Stress-induced
changes in vital signs also were heritable whether expressed
as maximal values, absolute changes (maximal minus basal),
or percent changes. Heritability was significant for baroreceptor slope, both upward BP deflections with reflex bradycardia (h2⫽33.3⫾9.2%; P⫽0.0004) and downward BP
deflections with reflex tachycardia (h 2 ⫽43.0⫾7.3%;
P⬍0.0001).
The h2 of traditionally heritable reference traits (weight at
h2⫽87⫾2%, P⬍0.0001; height at h2⫽93⫾1%, P⬍0.0001) in
our sample is consistent with previous observations.14
Twin TH Promoter Genotypes: Marker-on-Trait
Mapping/Associations
Univariate Analyses Across the Promoter
Significant associations were found for urinary catecholamine excretion (epinephrine and norepinephrine; Figure 2A)
and the BP response to environmental (cold) stress (⌬SBP
Circulation
August 28, 2007
A
Human tyrosine hydroxylase (TH):
SNP discovery in the local genomic region
5’-UTR
(19 nt)
TH
-1000
-1/+1
+1000
+3000
+4000
9
+5000
10 11 12
10 11
+6000
13
↑
TAG
12
C7461T/Asp467Asp (1.2%)
G7462A/Val468Met (0.3%)
C7535T/Ala492Val (0.3%)
G7550A/Gly497Asp (0.3%)
C7558T (0.3%)
G7796A (0.3%)
G7831A (0.6%)
7 8 9
7 8
C6449T/Arg410Trp (0.3%)
C6593T (1.7%)
C6594A (0.3%)
T6681C (40.5%)
6
C5975T (2%)
6
5
G5162C (27.7%)
A3034G (24%)
A3057G (0.6%)
G2426C (15.3%)
G2251A (0.3%)
+2000
4 5
4
T4120C (0.3%)
A4228T (0.6%)
C4253T (0.3%)
C4331A/Thr225Asn (0.6%)
3
T3832C (30.3%)
C3936A (0.6%)
3
2
T4581C (16.3%)
A4617G (0.6%)
G4779A/Lys240Lys (23.6%)
Position +/- ATG
2
1
G2066A/Val81Met (37.4%)
G2092A/Arg89Arg (0.6%)
(% minor allele frequency)
1
↑
ATG
C-833T (2..1%)
C-824T (35.5%)
G-814A (0.6%)
G-801C (11.5%)
T-741C (0.3%)
A-641G (1.8%)
A-581G (36.5%)
G-494A (47.6%)
C-388T (1.2%)
G-94T (0.3%)
G-18A (0.6%)
G16A/Ala6Thr (0.6%)
G44A/Arg15His (0.6%)
C50T/Ala17Val (0.3%)
G67A/Ala23Thr (0.3%)
G130A (0.3%)
G139A (0.3%)
G219A (5.3%)
(TCAT)n(74.5%het.) 671bp
Exons
Introns
G3236A/Ala143Thr (0.6%)
T H m RN A d o m a ins
SNPs
3’-UTR
(303 nt)
ORF
(1494 nt; 497 aa)
(TCAT)n
G1862A (27%)
A1893T (0.6%)
4
+7000
PCR amplicons
Resequencing (n=160 chromosomes) by Lian Zhang, Brinda K. Rana, and Daniel T O’Connor.
DTOC, 7-16-04.
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ORF (open reading frame)
UTR (untranslated region, 5’ or 3’)
Legend: UTR: untranslated region (5’ or 3’). ATG: initiation codon. nt: nucleotide. aa: amino acid. SNP: single nucleotide polymorphism. ORF: open reading frame.
(TCAT)n: tetranucleotide repeat in intron 1. Bp positions are numbered with respect to the ATG. Allele frequencies from n=80 individuals (160 chromosomes) in 4 ethnic groups.
B
Human tyrosine hydroxylase (TH) common polymorphisms
Exon/intron
TH
D’
Figure 1. Polymorphism discovery at TH. Polymorphism discovery in the local genomic region. SNP discovery in an ethnically diverse
sample (n⫽80 individuals; 2n⫽160 chromosomes) was studied. A, Resequencing strategy. Forty-nine SNPs and 1 tetranucleotide microsatellite were identified. Ten SNPs were located in 1-kbp proximal promoter, 14 in coding regions, and 25 in the untranslated regions
(UTRs) or in adjacent intronic regions. Of these, 13 SNPs were common (minor allele frequency ⱖ5%), including 4 in the TH proximal
promoter, 2 in coding regions, 4 in the UTRs, and 4 in intronic regions. Arrows indicate amplicons and the direction of resequencing. B,
Patterns of LD across the entire TH locus. Graphical overview of LD plot of point by-point LD among 14 polymorphisms with high
minor allele frequencies (13.6% to 42.6%, including the [TCAT]n in the first intron) spanning 7505 bp at the TH locus and proceeding
⬇1 kbp upstream (5⬘; promoter region). The white diagonal is the line of identity (y⫽x). The exon/intron structure of the TH locus is
shown at the top (note the lack of common polymorphisms in the final exon). The blue box in the lower left corner designates the LD
block at the proximal promoter. D’ is the LD parameter (scaled from 0 to 1). Results are from 2n⫽442 chromosomes in subjects of
European ancestry.
and ⌬DBP; Figure 2B) with variants at C-824T and A-581G.
Variants at G-801C and G-491A did not associate (Figure 2).
The common TH coding region/exon 2 nonsynonymous
variant Val81Met did not associate with catecholamines or
stress BP changes, nor did the less common (minor allele
frequency ⬍5%) coding region variants.
Multivariable Analyses Within the Promoter
Because both C-824T and A-581G seemed to influence
catecholamine secretion (Figure 2A) and the BP response to
stress (Figure 2B), we performed a multivariable analysis of
all 4 common TH promoter polymorphisms (C-824T,
G-801C, A-581G, and G-494A) in SOLAR in an attempt to
Rao et al
A
104
Norepinephrine 1/p
Epinephrine 1/p
p=0.0005
8.58%
Strength of association (1/p)
1000
100
p=0.0058
p=0.0069 5.57%
1.52%
p=0.0072
6.04%
p=0.0102
1.53%
← p=0.05
p=0.074
10
p=0.214
p=0.447
1
C-824T
B
10
4
Delta SBP 1/p
Delta DBP 1/p
p=0.0002
3.98%
5
Multiple Comparisons Within the TH Promoter
Because we tested the phenotypic effects of 4 different
promoter SNPs, we reevaluated the ␣ threshold to avoid
false-positive conclusions. The SNPSpD method of Nyholt18
tested intermarker correlations within the promoter block of
LD to yield an “effective” number of markers at 2.92,
indicating that an appropriate ␣ threshold to maintain the type
I error rate at ⱕ5% for a single phenotype would be
P⫽0.0171. If we include the common (⬇37%) coding region
(exon 2) polymorphism Val81Met in the SNPSpD analysis,
the effective number of markers rises to 3.73, and the
appropriate ␣ threshold falls to P⫽0.0134. The significance
levels for TH promoter polymorphism effects on catecholamine secretion and stress-induced BP changes exceeded
even these more stringent thresholds (Figure 2). We also
tested TH promoter SNPs against 2 general classes of
autonomic phenotypes, biochemical and physiological (Figure 2); because these traits are significantly correlated (online
Table I), a full Bonferroni correction would be inappropriately conservative22; alternatively, Sidak’s adjustment for
correlated traits would yield a revised ␣ threshold at
P⫽0.025323 for a single genotype. On the basis of the
principles of the less conservative false discovery rate,24 an
appropriate ␣ threshold for 2 phenotypic categories would be
P⫽0.0375.
1000
Strength of association (1/p)
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G-801C
G-494A
A-581G
Tyrosine hydroxylase promoter position
(bp upstream of ATG)
Functional Variants of TH
100
p=0.0087
1.53%
p=0.01
1.80%
p=0.026
1.07%
← p=0.05
10
p=0.182
p=0.781
p=0.199
p=0.667
1
C-824T
G-801C
A-581G
G-494A
Tyrosine hydroxylase promoter position
(bp upstream of ATG)
Figure 2. TH promoter polymorphisms. Region-specific prediction of autonomic traits in twins. A, Catecholamines. Univariate analyses across the promoter. Significance of association with catecholamine secretion graded by 1/P. The
probability value is derived from SOLAR of comparison of
individual TH promoter SNP in twins. The x axis indicates the
position (in bp upstream of ATG) of the common TH promoter
SNPs. Significance (horizontal black line) was set at P⬍0.05.
B, Stress BP. Univariate analyses across the promoter. Significance of association with change of BP response to cold
stress graded by 1/P. The probability value is derived from
SOLAR of comparison of individual TH promoter SNP in
twins. The x axis indicates the position (in bp upstream of
ATG) of the common TH promoter SNPs. Significance (horizontal black line) was set at P⬍0.05.
discern the most important variant(s). In this analysis,
C-824T became the most significant predictor of ⌬DBP
during cold stress (P⫽0.000164), although G-494A also
achieved significance (P⫽0.0276). We did not detect SNPby-SNP interactions (epistasis) within the TH promoter on
adrenergic traits (all P⬎0.2).
Permutation Tests
Another approach to multiple genotype–phenotype comparisons is the use of model-free exact (permutation) tests. We
dichotomized continuous traits about the median value and
then constructed 3⫻2 contingency tables (3 diploid genotypes by 2 trait quantiles). After permutation, the effect of
C-824T remained significant on both urinary norepinephrine
excretion (P⫽0.00633) and the DBP response to cold stress
(P⫽0.0157).
Sex
Because several traits differed by sex and age (online Table
II), we performed twin analyses on sex- and age-adjusted
traits within SOLAR or generalized estimating equations. We
also tested gene-by-sex interactions in marker-on-trait associations but did not find significant nonadditive interactions
for C-824T or A-581G on either catecholamines or stressinduced BP increments. We also tested each sex separately
for effects of TH promoter SNPs on these traits; significant
effects were seen for female subject (n⫽267) alone but not
for male subject alone (n⫽67), perhaps indicating a lack of
power to detect male-specific effects in the relatively small
number of male twins. Although California birth rates are
approximately equal for males and females, females are far more
likely to enroll in twin studies, especially younger females.25
Pleiotropy: Coordinate Effects of TH Promoter
C-824T on Biochemical and Physiological Traits
C-824T: Biochemistry and Physiology
C-824T was associated with urine catecholamine excretions
(epinephrine: P⫽0.0058; percent variation explained, 5.55%;
and norepinephrine: P⫽0.0069; percent variation, 1.52%).
The T-824C minor (T) allele was associated with higher
urinary catecholamine excretion (Figure 3A) and greater
6
Circulation
A
August 28, 2007
changes in BP response to cold stress (Figure 3B; ⌬SBP:
P⫽0.01; percent variation, 1.54%; and ⌬DBP: P⫽0.0069;
percent variation, 3.98%).
4
4 10
SOLAR (adjusted by age and sex):
Urine epinephrine: p=0.0058*, % variation = 5.55%
Urine nroepinephrine: p=0.0069*, % variation = 1.52%
4
Catecholamine excretion, ng/gm
3.5 10
C-824T: Pleiotropy (Norepinephrine and Stress BP)
C-824T exerted significant pleiotropic effects on the coupling
between BP response to cold stress and urinary norepinephrine (Figure 3C). Increasing T-824C minor (T) allele copy
number increased not only the change in DBP after cold
stress but also urinary norepinephrine excretion (bivariate
likelihood ratio analyses: ⌬DBP, ␹2⫽10.4, P⫽0.0013; ⌬SBP,
␹2⫽3.91, P⫽0.048). Similarly pleiotropic SBP results are
shown in online Figure II.
However, promoter SNP C-824T alone did not exert a
significant pleiotropic effect on the coupling between baroreceptor slope and norepinephrine secretion for either downward (bivariate ␹2⫽3.77, P⫽0.0522) or upward (bivariate
␹2⫽2.27, P⫽0.132) baroreceptor deflections (data not
shown).
33600
+/-1860
n=34
3 104
29000
+/-1440
n=146
2.5 104
26300
+/-1250
n=129
4
2 10
1.5 104
14600
+/-1130
n=34
1 104
12100
+/-500
n=146
11100
+/-520
n=129
5000
0
C-824T genotype
T/T
T/C
C/C
T/T
B
T/C
C/C
Norepinephrine
25
TH Promoter Haplotypes
SOLAR (adjusted by age and sex):
∆SBP: p=0.01*, % variation = 1.54%
∆DBP: p=0.0069*, % variation = 3.98%
From 10 SNPs in the TH proximal promoter, we inferred the
presence of 17 haplotypes; the 3 most common haplotypes
accounted for 84.8% of chromosomes examined (online
Table V). The most common haplotype overall (No. 1,
CCGGTAAACG) is rather frequent (⬎37.1%) in Asian,
Hispanic, and white populations but relatively unusual
(15.4%) in black samples. Haplotype 2 (CTGGTAGGCG) is
the most common variant in blacks (at 35.6%) but is relatively rare in Asians (10%). Haplotype 3 (CCGCTAAGCG)
is common in all (⬎11.6%) but blacks (7.7%). Similar
haplotypes were derived by considering only the 4 most
common promoter SNPs (C-824T, G-801C, A-581G, and
G-494A).
Patterns of LD across the TH promoter are illustrated in
online Figure III either across all ethnicities or in 4 discrete
ethnic/ancestry groups: European, African, Asian, and Hispanic. LD was substantial in the total group and population
subgroups, especially toward the 3⬘ end of the promoter. The
Asian and Hispanic groups displayed very high LD (D’ ⬎0.9)
across virtually the entire promoter region. White subjects
showed a falloff in LD toward the 5⬘ end of the region (D’
⬍0.4 at C-824T), whereas African ancestry subjects displayed especially low LD (D’ ⬍0.1) in the 5⬘ region with
preserved LD (D’ ⬎0.9) at the 3⬘ end of the domain.
Blood pressure change
after cold stress, mmHg
20
15
18.4+/-3.2
n=34
15.4+/-2.0
n=34
15.1+/-2.4
n=146
11.9+/-1.3
n=146
10
10.5+/-1.8
n=129
7.87+/-1.2
n=129
5
0
C-824T genotype
T/C
T/T
C/C
T/T
T/C
C/C
∆DBP
∆SBP
C
Change in DBP post cold stress, mmHg
Downloaded from http://circ.ahajournals.org/ by guest on June 17, 2017
Epinephrine
20
SOLAR (sex- and age-adjusted):
Urine norepinephrine: p=0.0069*, % variation = 1.52%
∆DBP: p=0.0002*, % variation = 3.98%
18
Bivariate: χ =10.4, p=0.0013*
2
16
T/T
n=34
14
12
C/T
n=146
10
8
C/C
n=129
6
4
2.4 10
4
2.6 10
4
2.8 10
4
3 10
4
4
3.2 10
3.4 10
4
4
3.6 10
Norepinephrine excretion, ng/gm
Figure 3. TH promoter polymorphism C-824T. Effects on catecholamine excretion and BP response to stress in twins. A,
Influence of TH promoter polymorphism C-824T on catecholamine excretion. Urinary epinephrine excretion: SOLAR
(adjusted by age, sex), P⫽0.0058, explaining 5.55% of trait variation; urinary norepinephrine excretion: SOLAR (adjusted by
age, sex), P⫽0.0069, explaining 1.52% of trait variation. B, Influence of TH promoter polymorphism C-824T on BP response to
stress. Change in SBP response to cold stress (⌬SBP): P⫽0.01,
explaining 1.54% of the trait variation; and change in DBP
response to cold stress (⌬DBP): P⫽0.0069, explaining 3.98% of
the trait variation. C, Pleiotropic effects of TH promoter polymorphism C-824T on the coupling between DBP response to
stress (⌬DBP) and urinary norepinephrine. Bivariate ␹2⫽10.4,
P⫽0.0013.
Rao et al
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Diploid Haplotypes (“Diplotypes”)
Haplotype 1/2 (CGAA/TGGG) diploid haplotype pairs influenced change in DBP during cold stress (⌬DBP: P⫽0.0312;
percent variation, 3.47%; online Table VIIA and Figure 4B).
Haplotype 1/3 (CGAA/CCAG) diploid combinations displayed associations with basal HR (P⫽0.0335; percent variation, 4.13%) and upward baroreceptor slope (P⫽0.0476;
percent variation, 4.49%; online Table VIIB). By contrast,
individual promoter SNPs did not predict baroreceptor slope
(all P⬎0.12).
TH Promoter Haplotypes and Pleiotropy: Joint
Effects on Biochemical and Physiological Traits:
Altering the Coupling Between Baroreceptor
Function and Efferent Sympathetic Outflow
Baroreceptor slopes (downward and upward deflections)
correlated highly with each other (␳⫽0.690, P⬍0.0001;
online Figure IVA). Urinary norepinephrine excretion inversely paralleled baroreceptor slopes, both upward
( ␳ ⫽⫺0.303, P⬍0.001; Figure 5A) and downward
(␳⫽⫺0.278, P⬍0.001; online Figure IVB) slope.
Not only did haplotype 2 (TGGG) display copy number–
dependent effects on urinary norepinephrine excretion
(P⫽0.0125; percent variation, 4.06%), but bivariate likelihood ratio analyses also indicated that TH haplotype 2 altered
the coupling of norepinephrine to baroreceptor activity for
both upward (␹2⫽8.0, P⫽0.0047; Figure 5A) and downward
(␹2⫽7.0, P⫽0.0082; online Figure IVB) deflection slope.
There were no univariate effects of haplotype 2 on baroreceptor downward deflection slope (P⫽0.702) or upward
deflection slope (P⫽0.497).
TH Promoter Haplotypes and Pleiotropy: Altering
the Coupling Between Norepinephrine Release and
Hemodynamic Responses to Environmental Stress
The effects of haplotype 2 (TGGG) displayed a pattern of
pleiotropy (Figure 5B). Increasing copy number of haplotype
15
16.3+/-2.1 16.9+/-3.1
n=164
n=32
14.9+/-2.0
n=32
12.4+/-1.1
n=164
10
10.7+/-1.8
n=131
8.17+/-1.2
n=131
5
0
Haplotype 2,
copies/genome
B
7
SOLAR (adjusted by age and sex):
∆SBP: p=0.0154*, % variation = 1.90
∆DBP: p=0.0004*, % variation = 3.73
20
Blood pressure change
during cold stress, mmHg
Haplotypes
We formed haplotypes from the 4 most common promoter
SNPs (C-824T, G-801C, A-581G, and G-494A) and then
tested whether haplotype copy number (0, 1, or 2/genome) at
the diploid locus influenced biochemical or physiological
trait means in twins (online Table VI). This approach is
feasible (ie, has sufficient power) for the most common
haplotypes in a population (here, haplotype 1 [CGAA], 2
[TGGG], or 3 [CCAG]).
The second-most-frequent promoter haplotype (haplotype
2, TGGG) displayed copy number– dependent effects on
stress change in BP (both ⌬SBP: P⫽0.0154; percent variation, 1.90%; and ⌬DBP: P⫽0.0004; percent variation, 3.73%;
Figure 4A) and urinary epinephrine (P⫽0.0044; percent
variation, 5.7%) and norepinephrine (P⫽0.0125; percent
variation, 4.06%) excretion (online Table VI). Haplotype 3
(CCAG) displayed associations with both basal heart rate and
change in heart rate during cold stress (P⫽0.0104; percent
variation, 3.14%; online Table VI). However, there were no
associations for trait means with copy number of the most
frequent haplotype (haplotype 1, CGAA).
A
0
1
2
∆SBP
0
1
2
∆DBP
20
SOLAR (adjusted for age & sex):
p=0.0312*, % variation = 3.47
Diastolic blood pressure change
during cold stress, mmHg
TH Promoter Haplotype Effects on
Autonomic Traits
Functional Variants of TH
15
15.1+/-2.01
n=32
12.1+/-1.32
n=121
10
9.7+/-1.47
n=79
5
0
Hap1/Hap1
Hap1/Hap2
Hap2/Hap2
Tyrosine hydroxylase promoter diploid haplotype
Figure 4. TH promoter haplotypes and autonomic traits. A,
Influence of TH promoter haplotype 2 (TGGG) on BP response
to stress. Change in systolic BP response to cold stress (⌬SBP):
P⫽0.0154, explaining 1.90% of the trait variation; and DBP
response to cold stress (⌬DBP): P⫽0.0004, explaining 3.73% of
the trait variation. B, Diploid haplotypes 1 and 2. Influence of TH
promoter haplotypes 1 and 2 on change in DBP response during cold stress: P⫽0.0312, explaining 3.47% of the trait
variation.
2 augmented changes in DBP (P⫽0.0004; 3.73% variation
explained) during cold stress and urinary norepinephrine
excretion (P⫽0.0125; 4.06% variation explained), and the
likelihood ratio test indicated a coordinate effect on the 2
traits (bivariate ␹2⫽14.2, P⫽0.0002).
TH Promoter Haplotype Lineage and Coalescence
Human phylogeny at the TH promoter was approached using
haplotypes imputed from the 4 common (high minor allele
frequency) SNPs in the proximal promoter (C-824T, G-801C,
A-581G, and G-494A). Coalescent simulations (Figure 6A)
provide a graphical representation of the most likely ancestry of
the disease-associated haplotypes in recent human evolutionary
8
Circulation
August 28, 2007
A
2
4 10
Norepinephrine h =49.6+/-6.7%, p=0.0001*
Haplotype 2 on norepinephrine: p=0.0125*,
4.06% variation explained
4
2
Urinary norepinephrine, ng/gm
Baroreceptor upward slope h =33.3+/-9.2%, p=0.0004*
Haplotype 2 on baroreceptor upward slope: p=0.497
Pleiotropy: bivariate likelihood ratio test
2
χ =8.0, p=0.0047*
4
3.5 10
Haplotype 2
n=2 copies
(n=32 individuals)
3 10 4
Haplotype 2
n=1 copy
(n=164 individuals)
Haplotype 2
n=0 copies
(n=131 individuals)
4
2.5 10
2 104
10
5
15
20
Baroreceptor upward deflection slope, msec/mmHg
B
18
2
Norepinephrine h =49.6+/-6.7%, p=0.0001*
Haplotype 2 on norepinephrine: p=0.0125*,
4.06% variation explained
2
Change in DBP during cold stress, mmHg
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Upward deflections.
16
∆DBP h =32+/-8%, p=0.0003*
Haplotype 2 on ∆DBP: p=0.0004*,
3.73% variation explained
Pleiotropy: bivariate likelihood ratio test
2
χ =14.2, p=0.0002*
Haplotype 2
n=2 copies
(n=32 individuals)
14
12
Haplotype 2
n=1 copy
(n=164 individuals)
10
8
Haplotype 2
n=0 copies
(n=131 individuals)
6
4
2.4 10
2.6 104
2.8 10
4
4
3 10
3.2 10
4
3.4 10
4
3.6 10
4
Norepinephrine excretion, ng/gm
Figure 5. TH promoter haplotypes and pleiotropy. A, Baroreceptor control of efferent sympathetic outflow. Baroreceptor upward deflections and sympathetic outflow. Left, Negative effect of renal norepinephrine excretion on baroreceptor slope (upward deflections).
Spearman ␳⫽⫺0.303, P⬍0.001. Right, Pleiotropic effect of TH promoter haplotype 2 on the coupling between the baroreflex (upward
deflections) and catecholamine secretion. Bivariate ␹2⫽8.0, P⫽0.0047. B, Sympathetic outflow and the hemodynamic response to environmental stress (DBP). Pleiotropic effect of TH promoter haplotype 2 on the coupling between change in DBP during cold stress
(⌬DBP) and renal norepinephrine excretion. Bivariate ␹2⫽14.2, P⫽0.0002.
history.16 The most common haplotype (haplotype 1, CGAA)
seemed to arise by point mutation from its most likely ancestral
haplotype, CGGG, corresponding to an A-581G purine/purine
transition ⬇224 000 years ago. The second most common
haplotype (haplotype 2, TGGG) likely arose by point mutation
from its most recent common ancestor, CGGG, corresponding to
a C-824T pyrimidine/pyrimidine transition ⬇381 000 years ago.
Of note, haplotype 2, which is associated with greater BP and
catecholamines (Figures 4 and 5), is the most common haplotype in subjects of African ancestry (online Table V).
TH Promoter Variants: Functional
Consequences Probed by Chromaffin Cell
Transfection/Expression
We assayed haplotype-specific gene expression in PC12
chromaffin cells (Figure 6B) with TH promoter/luciferase
Rao et al
9
Ancestral (chimp) haplotype
B
↑
S t ro n ge r
9
3 10
2.5 10
1
CGAA
3
CGAG
2
CCAG
CGGG
Chromosomes: n=
Haplotype #: 8
Norepinephrine renal excretion in vivo
(ng/gm creatinine)
Kilo-years ago
3.5 10
TGGG
Time
A
Functional Variants of TH
↑
Weaker
2 10
4
Promoter haplotype #2
-824T → -581G
Homozygotes (n=17)
Stronger
ha p l ot y pe :
TGGG
4
4
Promoter haplotype #1
C-824 → A-581
Homozygotes (n=33)
Weaker
ha pl o ty p e :
CGAA
In vivo results in twins
(norepinephrine excretion in haplotype homozygotes)
Norepinephrine excretion: T=3.43, p=0.0012*
Promoter strength: T=3.00, p=0.04*
4
0.3
0.32
0.34
0.36
0.38
0.4
Tyrosine hydroxylase promoter strength in chromaffin cells in vitro :
transfected TH promoter haplotype → luciferase reporter (firefly/renilla)
Downloaded from http://circ.ahajournals.org/ by guest on June 17, 2017
Figure 6. TH promoter haplotypes. Phylogeny and function. A, Phylogeny. Coalescent simulation of likely lineages in human history.
From the 4 common (minor allele frequency ⬎10%) TH promoter SNPs (C-824T, G-801C, A-581G, G-494A), PHASE imputed 17 haplotypes, the 5 most common of which are shown here. In this coalescent, the transcriptionally weaker haplotype, CGAA (haplotype 1),
seemed to arise by point mutation from its most likely ancestral haplotype, CGGG, corresponding to an A-581G purine/purine transition
⬇224 000 years ago. The transcriptionally stronger haplotype, TGGG (haplotype 2), seemed to arise by point mutation from its most
recent common ancestor, CGGG, corresponding to a C-824T pyrimidine/pyrimidine transition ⬇381 000 years ago. The ultimate ancestral haplotype was specified as CGGG on the basis of the alleles in both chimpanzee DNA and the major alleles in an ancestral human
population (African ancestry). B, Function. Coordinate effects on in vitro TH transcription and in vivo catecholamine production. Results
from promoter haplotype reporter transfection into PC12 chromaffin cells in vitro are shown on the x axis, whereas renal norepinephrine
excretion in vivo results for haplotype homozygotes are shown on the y axis. Haplotype 1 (CGAA) displays weaker promoter strength in
vitro, whereas CGAA/CGAA homozygotes had diminished renal norepinephrine secretion in vivo. Conversely, haplotype 2 (TGGG)
showed increased activity both in vitro and in vivo.
reporters for the 2 most common promoter haplotypes.
Haplotype 2 (TGGG) was substantially more active in
chromaffin cells; Figure 6B illustrates the increased activities of haplotype 2 both in vitro (driving transcription) and
in vivo (determining norepinephrine secretion).
Relatively low-expressing haplotype 1 (CGAA), is the
most common variant in Asian (61.7%), white (46.8%), and
Hispanic (37.1%) populations but is relatively unusual
(15.4%) in blacks. The higher-expressing haplotype 2 is the
most common variant in blacks (34.6%).
TH Promoter and Disease: Hypertension
Figure 7 illustrates a population-based case-control study in
which ⬎1200 subjects were drawn from the highest and
lowest fifth percentiles of BP in a primary care practice of
⬎53 000 adults. In a 2-way ANOVA, there was a significant
sex-by-genotype (TH C-824T) interaction on DBP
(P⫽0.044). As expected, sex also influenced DBP
(P⬍0.001). When the ANOVA was run in the presence or
absence of C-824T, the results indicated that C-824T variation accounted for 3.4% of population DBP variance. When
analyses were conducted separately on the sexes, the C-824T
genotype effect was found in male subjects (P⫽0.045) but
not female subjects (P⫽0.985). Inspection of the bar graph
indicates that increasing numbers of the minor (T) allele
increased DBP in male but not female subjects.
To explore the findings in the same group in a model-free
fashion, without relying on standard asymptotic assumptions,
we also used more computationally intensive permutation
tests. We dichotomized subjects by BP status (high versus
low) and sex (male versus female). We then conducted 3⫻2
table permutation tests on the effect of C-824T diploid
genotype (C/C, C/T, or T/T) on BP status; the effect remained
significant in male (P⫽0.023) but not female (P⫽0.266)
subject.
To replicate the TH effects on BP in an independent
sample, we studied the C-824T polymorphism in 898 additional subjects, but this time not from population BP extremes: 352 with hypertension and 546 with normal BP. Once
again, the C-824T affected DBP (F⫽7.73, P⬍0.001; Figure
7B), although without a gene-by-sex interaction in these
subjects with less extreme BPs. If we adjusted DBP data for
the effects of antihypertensive medications,26 the effect of
C-824T on DBP persisted (F⫽7.88, P⬍0.001). Finally,
C-824T also affected SBP (F⫽5.61, P⫽0.004).
Discussion
Overview
Critical Role of the Enzyme TH in
Catecholamine Metabolism
TH
catalyzes
the
conversion
of
tyrosine
to
L-dihydroxyphenylalanine, the rate-limiting step in the biosynthesis of catecholamines.1,27 Profound TH deficiency, as
occurs after unusual inactivating mutations (Leu205Pro or
Gln381Lys) in homozygous individuals, results in widespread disturbance of neuropsychiatric function such as
autosomal-recessive, L-dihydroxyphenylalanine–responsive
dystonia.27 Complete homozygous ablation of the TH locus
August 28, 2007
by homologous recombination-directed gene targeting in
transgenic mice is lethal by the early postnatal period.28
Previous Work: (TCAT)n Intronic Polymorphism at TH
Differential allele frequencies for the intronic (TCAT)n microsatellite polymorphism have been associated with hypertension4 and BP regulation.8,29 However, the functional significance of the (TCAT)n polymorphism remains in doubt.8,9
Therefore, we systematically searched the TH locus for a
functional polymorphism.
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Comprehensive TH SNP Discovery and Role of
Polymorphism in the TH Coding Region
Does common qualitative (amino acid changing) polymorphism in this gene contribute to variation in autonomic tone?
In the coding region, we found 2 common biallelic variants,
only one of which was nonsynonymous: Val81Met, at 37.4%
(Figure 1A), a polymorphism of uncertain significance, lying
outside the catalytic domain,30 which did not associate with
autonomic traits. We also found 12 unusual nonsynonymous
coding region variants (Figure 1A), but their allele frequencies were only 0.3% to 0.6%, not sufficient to account for
population associations. Thus, we turned to potential regulatory (noncoding) variants.
Comprehensive Polymorphism Discovery in the
TH Promoter
Visual inspection (Figure 1B) of GOLD plots of LD structure
across TH reveals 2 blocks of particularly high LD: at the 5⬘
(promoter) and the mid to 3⬘ regions of the gene. Thus, we
examined the promoter block for functional consequences.
We found 10 SNPs in the proximal promoter (online Table I),
4 of which were common and resolved themselves into 4
common haplotypes (online Table V).
A
100
95
90
2-way ANOVA:
(Covariates: age, BMI)
Overall F=12.4, p<0.001*
Genotype F=1.30, p=0.273
Sex F=31.6, p<0.001*
Genotype * Sex F=3.14, p=0.044*
C-824T explains 3.4% of DBP variance
Alleles: C=61%, T=39%
Male DBP
Female DBP
Males alone:
Genotype F=3.12, p=0.045*
Females alone:
Genotype F=0.015, p=0.985
2
HWE: χ =0.54, p=0.46
DBP, mmHg
(mean +/- SEM)
Circulation
85
75
85.3
+/-2.6
(n=75)
82.8
+/-1.3
(n=285)
80
78.3
+/-1.5
(n=234)
70
74.9
+/-1.5
(n=233)
74.9
+/-1.2
(n=329)
73.2
+/-2.1
(n=110)
65
60
C/C
C/T
T/T
Tyrosine hydroxylase (TH) promoter C-824T diploid genotype
B
85
2-way ANOVA:
Genotype F=7,73, p<0.001
Sex F=45.2, p<0.001
Genotype-by-Sex F=0.66, p=0.518
Males
Females
80
DBP, mmHg
10
305
168
75
227
57
80
70
TH Promoter Variants and Autonomic
Pathway Pleiotropy
Because the same TH promoter SNPs predicted both biochemical (Figures 2A and 3A) and physiological (Figure 3A
and 3B) traits, we undertook bivariate genetic analyses15 in
search of pleiotropy or the coordinate effect of a single gene
on multiple traits. Bivariate results indicate coupled genetic
control of both catecholamine secretion and stress BP by
C-824T (Figure 3C). Pleiotropy extended into haplotypic
control of both catecholamine secretion and baroreceptor
function (Figures 4 and 5).
Multivariable Promoter Analyses
Because both C-824T and A-581G influenced both catecholamine secretion (Figure 2A) and BP response to stress
(Figure 2B), we performed a multivariate analysis of all 4
common TH promoter polymorphisms to discern important
variant(s). In this analysis, C-824T became the most significant predictor of change in DBP during cold stress
(P⫽0.000164); G-494A also retained significance
(P⫽0.0276).
Dating the Responsible TH Promoter Variants
We approached likely dates of origin of trait-associated TH
promoter variants using the coalescent approach. 16
A-581G likely arose ⬇224 000 years ago; C-824T was
even more ancient, arising ⬇381 000 years ago (Figure
90
65
C/C
C/T
T/T
Tyrosine hydroxylase (TH) promoter C-824T diploid genotype
Figure 7. TH promoter polymorphism and hypertension. A, Population BP extremes. C-824T genotype interacts with sex to
determine BP in the population. The sample constituted ⬎1200
individuals selected from the highest and lowest fourth to fifth
percentiles of DBP in a primary care (Kaiser) population of
⬎53 000. By 2-way ANOVA, there was a significant genotypeby-sex interaction (F⫽3.14, P⫽0.044), with results most prominent in males. Males alone also had a significant effect of genotype on DBP (F⫽3.12, P⫽0.045). B, Replication in subjects with
less extreme BPs. This independent replication sample (from
University of California San Diego/San Diego VA) included 352
subjects with essential hypertension and 546 with normal BP.
Here, the effect of C-8224T on DBP also was significant
(F⫽7.73, P⬍0.001), but there was no gene-by-sex interaction
(F⫽0.66, P⫽0.518).
6A). What is the significance of such an ancient origin and
modern persistence at high frequency (eg, ⫺824T at
⬇17% to 60%; online Table I) of such alleles? Of note, the
⫺824T allele is associated with increased catecholamine
production (Figure 3A), increased BP increments in response to stress (Figure 3B), and extreme BP values in the
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Sex, TH Polymorphism, and BP
Twin data indicate that autonomic function differs between
males and females in both BP and catecholamine secretion
(online Table II). Sex differences in plasma epinephrine
(female less than male) confirm our previous observations,14
whereas the sex differences in urinary epinephrine and
norepinephrine (also female less than male) are novel. The
most striking sex effect we noted was on TH polymorphism
(C-824T) and population BP extremes (Figure 7); here, a
gene-by-sex interaction was apparent (interaction F⫽3.14,
P⫽0.044) whereby the polymorphism had affected BP in
males (F⫽3.12, P⫽0.045) but not females (F⫽0.015,
P⫽0.985). This gene-by-sex interaction may be a clue to
genetic mechanisms underlying the well-known and substantial differences in adrenergic function between men and
women.31 Of note, although a gene-by-sex interaction on BP
was found in population BP extremes (Figure 7A), C-824T
seemed to affect DBP in both male and female subjects in a
sample of hypertensives and normotensives with less extreme
BPs (F⫽7.73, P⬍0.001; Figure 7B).
Ethnicity and TH Polymorphism
TH allele (online Table I) and haplotype (online Table V)
frequencies differed by ethnicity, with subjects of African
ancestry displaying the most striking differences. In particular, the TH ⫺824T allele and its associated haplotype 2
(TGGG) were especially frequent in blacks; ⫺824T became
the quantitatively major allele (at 57%; online Table I) and
TGGG the most frequent haplotype (at 34.6%; online Table
V). In white subjects, the ⫺824T allele and haplotype 2
predicted greater catecholamine production (Figure 3A),
stress BP increments (Figure 3B), and BP in the population
(Figure 7). Because blacks have a greater population prevalence of hypertension32 and adrenergic reactivity to stress,33,34
we speculate that TH polymorphisms might be involved in
such traits with disproportionate frequency across ethnic
populations. However, explicit association tests of these TH
variants with disease or physiology in subjects of African
ancestry remain to be done.
Study Strengths: Coupling the Twin Method and
Systematic Polymorphism Discovery With
Adrenergic Phenotyping
Twin Phenotyping Protocol
We exploited the classic twin design.35,36 Twin data offer the
advantage of heritability (h2) measurement, the fraction of
phenotypic variance accounted for by genetic variance, a
logical estimator of the tractability of any trait to genetic
investigation. Because twins are randomly sampled from the
population, genetic conclusions drawn from twin studies are
likely to be generalizable to the population from which they
were sampled.35 Multiple autonomic phenotypes in the twins,
both biochemical and physiological, allowed construction of
an integrated picture of the effects of particular genetic
variants at TH (Figure 8).
Functional Variants of TH
Concept:
Application to TH:
Gene
↓
Biochemical trait
↓
Physiological trait(s)
Tyrosine hydroxylase (TH) C-824T
↓
↑ Catecholamines
↓
↓ B a r o r e c e p t or f u n c t i o n
↓
↑ Stress blood pressure
↓
↑ Hypertension
Disease trait
11
Mechanism
population (Figure 7). We speculate that the functional
variation we observed in ⫺824T carriers could be the
outcome of environmental selective pressures acting on
alleles augmenting catecholaminergic function.
Time
Rao et al
Figure 8. Intermediate phenotypes and blood pressure. The
“intermediate phenotype” viewpoint formulates a series of timedependent phenotypic manifestations of complex trait genetic
variants; biochemical traits are hypothesized to be determined
directly by genotype, followed later by physiological traits and
ultimately by late-penetrance disease (such as hypertension).
Here, the concept is illustrated by the findings at TH in the present study: the TH promoter variant C-824T initially alters norepinephrine production, subsequently influencing baroreceptor
slope and transient stress BP responses (ie, gene-byenvironment interaction), finally predisposing to the development
of fixed/established hypertension in the population.
Systematic Polymorphism Discovery
LD mapping is an increasingly powerful tool for exploring
genetic determinants of disease.37 However, the LD approach
requires fulfillment of many assumptions,38 including substantial LD between marker and trait alleles. Here, we took
another approach: systematic polymorphism discovery at a
candidate genetic locus. This approach enables direct testing
of marker-on-trait allelic association rather than indirect
testing relying on a hypothetical degree of LD between
marker and trait alleles. Of note, at the TH locus, we
discovered 2 blocks of LD (Figure 1B) and found that the
SNP most commonly used in previous LD studies at TH,
Val81Met (G2066A, exon 2), was in a different (downstream) “block” of LD from the promoter (upstream) block;
of further note, Val81Met itself did not associate with
autonomic traits, establishing the necessity of systematic SNP
discovery as a prerequisite to effective exploration of the
functional consequences of polymorphism at the TH locus.
SNPs and Haplotypes
Dense promoter genotyping (by systematic resequencing) in a
large series of twins permitted both individual SNP (Figures
2 and 3) and haplotype (online Table VI and Figure 4)
approaches to trait associations. In the presence of already
complete genomic information at the TH proximal promoter
(online Table I and Figure 1), haplotypes might not provide
any new associations by virtue of LD; indeed, we derived
SNP genotypes in the TH promoter by resequencing that
region in 172 twin pairs. Twin analyses in SOLAR allowed us
to quantify the contribution of each polymorphism (single
SNP or haplotype) to the adrenergic traits in the form of
percent of trait variance explained. In general, trait predictions by individual SNPs (Figures 2 and 3) and haplotypes
(online Table VI and Figure 4) were comparable. However,
several observations here suggest that ⬎1 variant in the TH
promoter may be important for trait determination: (1) Two
strong univariate SNP-on-trait associations were found in the
TH promoter at both C-824T and A-581G (Figures 2 and 3);
(2) a multivariable analysis of the TH promoter indicated that
⬎1 SNP (both C-824T and G-494A) influenced stress BP
12
Circulation
August 28, 2007
responses; and (3) haplotypes, but not individual SNPs,
predicted baroreceptor slope (Table IIIB) and its pleiotropy
with catecholamine secretion (Figure 5A). In the population,
however, only a single TH SNP (C-824T) predicted BP
extremes (Figure 7). Diploid haplotype combinations (pairs)
also predicted adrenergic traits (online Table VII and Figure
4); in general, pairs were not more predictive than individual
haplotype copy numbers (0, 1, 2 copies).
Downloaded from http://circ.ahajournals.org/ by guest on June 17, 2017
Ultimate Disease State Association: Hypertension
To establish the pertinence of our observations for human
disease, we also genotyped individuals with extreme of BP as
a quantitative trait in a population-based cohort (Figure 7A).
Indeed, common TH promoter variation at C-824T accounted
for up to ⬇3.4% of the population DBP variance, and the
effect was replicated (Figure 7B). These results document the
“intermediate phenotype”39,40 approach as a successful route
to discovery of genetic variants underlying a complex disease
trait.
Complex (Nonmendelian) Inheritance
“Intermediate” Phenotypes, Pathways, and Pleiotropy
Investigations of putative pathways toward disease (Figure 8)
yielded coordinate or pleiotropic effects of TH promoter
variants on both biochemical and physiological traits (Figures
3, 5, and 8). We documented such pleiotropy statistically
using bivariate genetic analyses.15 Genetic pleiotropy is an
integral component of the “intermediate phenotype” hypothesis (Figure 8), wherein 1 gene influences a series of traits
over time.
Gene-by-Environment Interactions
In addition to TH effects on resting traits (Figure 2A), TH
variation also predicted the BP response to cold stress (Figure
2B). This is a classic example of a gene-by-environment
interaction,41,42 requiring both a specific genetic variant (here,
C-824T) and an environmental perturbation (here, cold) for
expression of the trait (in this case, ⌬BP).
TH and Sex
Sex had a substantial effect on many of the adrenergic
intermediate phenotypes we evaluated (online Table II). In
addition, sex seemed to play a permissive role for the action
of the TH C-824T genotype on individuals with the most
extreme (highest and lowest) BP in the population (Figure
7A), but in people with less extreme BP values, the genotype
affected DBP in both males and females (Figure 7B). We do
not precisely understand the nature of this TH gene-by-sex
interaction, although it may be rooted in the different hormonal milieu subserving autonomic and cardiovascular function in males and females.31 Fundamental molecular and
cellular mechanisms of BP control may differ in males and
females,31 but the ultimate implications of such differences
for disease states such as hypertension are not clearly understood. Indeed, we have noted that aging-dependent changes in
sympathetic activity differ between the sexes,43 as do vascular
responses to adrenergic agonists.44 The C-824T genotype-bysex interaction has potential implications. Since sex may
modify the C-824T effect on BP, sex may contribute to the
role of genotype in the diagnosis, pathogenesis, or treatment
of hypertension.
Functional Documentation in Chromaffin Cells
What is the mechanism by which TH 5⬘ allelic variants
(Figure 1A and online Table I) influence human autonomic
traits? We tested differential regulation of TH transcription
with 1155-bp promoter haplotype/reporter plasmids transfected and expressed in chromaffin cells (Figure 6B). Indeed,
we confirmed functional differences between TH promoter
variants in vitro (Figure 6B), and the differences paralleled
associations of these same variants with catecholamine secretion in vivo (Figure 5B). Thus, the 4 TH variants under
consideration clearly lie in a domain with transcriptional
activity, altering TH promoter strength in vitro.
Study Limitations and Caveats
Complex Trait Genetics
Multiple alleles may yield multiple traits. Genetic analyses of
a complex trait necessitate the consideration of multiple
phenotypes and genotypes, raising the possibility of falsepositive (type I) statistical errors. We approached this issue in
several ways: haplotyping, pleiotropic/bivariate analyses (1
gene yields ⬎1 trait), multivariable analyses (⬎1 SNP yields
1 trait), SNPSpD (determining the “effective” number of
SNPs within a block of LD and thereby adjusting the required
threshold for significance of a single phenotype), permutation
(exact) tests, and finally replication. TH haplotypes, simultaneously considering each common variant within the promoter, predicted autonomic traits (Figures 4 and 5). A
multivariable analysis established the particular role of
C-824T on BP. SNPSpD determined that the promoter SNP
effects on single autonomic traits exceeded chance alone.
Permutation established an empirical level of significance;
the effects of C-824T on twin traits and BP in the population
remained significant. Finally, replication established the effect of C-824T on hypertension.
Haplotype Assignment Uncertainty
Imputation of phase from diploid genotype data is inherently
uncertain and occasionally prone to misclassification; the
haplotype method we used assigns the 2 most likely haplotypes to each individual.45 Although emerging haplotype
methods consider uncertainty in phasing,46,47 such methods
have not yet been coupled with the computational needs of
correlated twin pair statistics.
Ethnicity
Although we conducted systematic variant discovery in both
black and white subjects, the studies on autonomic physiology were analyzed only in white (European ancestry) subjects, initially to avoid the potentially spurious effects of
population stratification on genetic trait associations.48 Only
additional studies can determine whether the associations in
white subjects are generalizable to other population groups.
Conclusions
We conclude that catecholamine secretory traits are heritable
(online Table IV), displaying joint genetic determination (or
pleiotropy) (Figure 5) with autonomic activity and finally
with BP in the population (Figures 7 and 8). Interindividual
Rao et al
differences in catecholamine secretion are influenced by
genetic variation in the adrenergic pathway encoding catecholamine synthesis, especially at the classically rate-limiting
step, TH. These results document novel pathophysiological
links between a key adrenergic locus, catecholamine metabolism, and BP (Figure 8) and suggest new strategies to
approach the mechanism, diagnosis, and treatment of systemic hypertension.
Hypothesis Schematic
A schematic formulating our results into a global hypothesis
is presented in Figure 8 and outlines the role of intermediate
phenotypes and the TH candidate gene (especially its C-824T
promoter variant) in the determination of hypertension via the
initial intermediary of catecholamine metabolism.
Downloaded from http://circ.ahajournals.org/ by guest on June 17, 2017
Implications for Pathophysiology/Mechanism,
Prediction/Diagnosis, and Treatment
Our results suggest that the adrenergic pathway is centrally
involved in the early pathogenesis of hypertension beginning
in healthy individuals, perhaps initially by altering baroreceptor function (Figures 5 and 8) or consequently the transient
response BP response to environmental stress (Figures 2, 3,
and 8). Adrenergic genetic determination of biochemical
(Figures 2A and 3A) and physiological (Figures 2B and 3B)
traits and the ultimate disease trait (Figure 7) suggests that
treatments targeting the adrenergic pathway might be beneficial in preventing hypertension if administered to subjects at
specific genetic risk. Our results also raise the possibility that
adrenergic genetic profiling of patients with impaired autonomic activity or increased stress BPs might yield practical
pharmacogenetic predictors of patients most likely to benefit
from sympatholytic therapy.
Future Directions/Studies: Implications for Prevention:
Heredity and Environment
Our results raise the possibility that profiling subjects for
particular adrenergic and signaling polymorphisms would
provide an index of risk for or susceptibility to hypertension.
This prediction awaits testing in appropriate longitudinal
cohorts.
Acknowledgments
The authors appreciate the assistance of the General Clinical Research Center (RR00827) and its core laboratory.
Sources of Funding
This work was supported by the Department of Veterans Affairs and
the National Institutes of Health, Bethesda, Md.
Disclosures
None.
References
1. Flatmark T, Stevens RC. Structural insight into the aromatic amino acid
hydroxylases and their disease-related mutant forms. Chem Rev. 1999;
99:2137–2160.
2. Kobayashi K, Nagatsu T. Molecular genetics of tyrosine 3-monooxygenase and inherited diseases. Biochem Biophys Res Commun. 2005;338:
267–270.
3. Zhou QY, Quaife CJ, Palmiter RD. Targeted disruption of the tyrosine
hydroxylase gene reveals that catecholamines are required for mouse fetal
development. Nature. 1995;374:640 – 643.
Functional Variants of TH
13
4. Sharma P, Hingorani A, Jia H, Ashby M, Hopper R, Clayton D, Brown
MJ. Positive association of tyrosine hydroxylase microsatellite marker to
essential hypertension. Hypertension. 1998;32:676 – 682.
5. Lim LC, Gurling H, Curtis D, Brynjolfsson J, Petursson H, Gill M.
Linkage between tyrosine hydroxylase gene and affective disorder cannot
be excluded in two of six pedigrees. Am J Med Genet. 1993;48:223–228.
6. Meloni R, Laurent C, Campion D, Ben Hadjali B, Thibaut F, Dollfus S,
Petit M, Samolyk D, Martinez M, Poirier MF, Mallet J. A rare allele of
a microsatellite located in the tyrosine hydroxylase gene found in schizophrenic patients. C R Acad Sci III. 1995;318:803– 809.
7. Thibaut F, Ribeyre JM, Dourmap N, Meloni R, Laurent C, Campion D,
Menard JF, Dollfus S, Mallet J, Petit M. Association of DNA polymorphism in the first intron of the tyrosine hydroxylase gene with disturbances of the catecholaminergic system in schizophrenia. Schizophr
Res. 1997;23:259 –264.
8. Zhang L, Rao F, Wessel J, Kennedy BP, Rana BK, Taupenot L, Lillie EO,
Cockburn M, Schork NJ, Ziegler MG, O’Connor DT. Functional allelic
heterogeneity and pleiotropy of a repeat polymorphism in tyrosine
hydroxylase: prediction of catecholamines and response to stress in twins.
Physiol Genomics. 2004;19:277–291.
9. Albanese V, Biguet NF, Kiefer H, Bayard E, Mallet J, Meloni R. Quantitative effects on gene silencing by allelic variation at a tetranucleotide
microsatellite. Hum Mol Genet. 2001;10:1785–1792.
10. Do KA, Broom BM, Kuhnert P, Duffy DL, Todorov AA, Treloar SA,
Martin NG. Genetic analysis of the age at menopause by using estimating
equations and Bayesian random effects models. Stat Med. 2000;19:
1217–1235.
11. Almasy L, Blangero J. Multipoint quantitative-trait linkage analysis in
general pedigrees. Am J Hum Genet. 1998;62:1198 –1211.
12. Stephens M, Smith NJ, Donnelly P. A new statistical method for haplotype reconstruction from population data. Am J Hum Genet. 2001;68:
978 –989.
13. Abecasis GR, Cookson WO. GOLD: graphical overview of linkage disequilibrium. Bioinformatics. 2000;16:182–183.
14. Falconer DS, Mackay TFC. Heritability. In: Introduction to Quantitative
Genetics. Essex, England: Longman; 1996;160 –183.
15. Almasy L, Dyer TD, Blangero J. Bivariate quantitative trait linkage
analysis: pleiotropy versus co-incident linkages. Genet Epidemiol. 1997;
14:953–958.
16. Rosenberg NA, Nordborg M. Genealogical trees, coalescent theory and
the analysis of genetic polymorphisms. Nat Rev Genet. 2002;3:380 –390.
17. Bahlo M, Griffiths RC. Inference from gene trees in a subdivided population. Theor Popul Biol. 2000;57:79 –95.
18. Nyholt DR. A simple correction for multiple testing for single-nucleotide
polymorphisms in linkage disequilibrium with each other. Am J Hum
Genet. 2004;74:765–769.
19. Mehta CR, Patel NR. A network algorithm for performing Fisher’s exact
test in RxC contingency tables. J Am Stat Assoc. 1983;78:427– 434.
20. Clarkson D, Fan YA, Joe H. A remark on algorithm 643: FEXACT: an
algorithm for performing Fisher’s exact test in RxC contingency tables.
ACM Trans Mathematical Software. 1993;19:484 – 488.
21. Gonzalez-Trapaga JL, Nelesen RA, Dimsdale JE, Mills PJ, Kennedy B,
Parmer RJ, Ziegler MG. Plasma epinephrine levels in hypertension and
across gender and ethnicity. Life Sci. 2000;66:2383–2392.
22. Perneger TV. What’s wrong with Bonferroni adjustments. BMJ. 1998;
316:1236 –128.
23. Sankoh AJ, Huque MF, Dubey SD. Some comments on frequently used
multiple endpoint adjustment methods in clinical trials. Stat Med. 1997;
16:2529 –2542.
24. Benjamini Y, Yekutieli D. The control of the false discovery rate in
multiple testing under dependency. Ann Stat. 2001;29:1165–1188.
25. Cockburn M, Hamilton A, Zadnick J, Cozen W, Mack TM. The
occurrence of chronic disease and other conditions in a large
population-based cohort of native Californian twins. Twin Res. 2002;5:
460 – 467.
26. Cui JS, Hopper JL, Harrap SB. Antihypertensive treatments obscure
familial contributions to blood pressure variation. Hypertension. 2003;
41:207–210.
27. Flatmark T. Catecholamine biosynthesis and physiological regulation in
neuroendocrine cells. Acta Physiol Scand. 2000;168:1–17.
28. Carson RP, Robertson D. Genetic manipulation of noradrenergic neurons.
J Pharmacol Exp Ther. 2002;301:410 – 417.
29. Barbeau P, Litaker MS, Jackson RW, Treiber FA. A tyrosine hydroxylase
microsatellite and hemodynamic response to stress in a multi-ethnic
sample of youth. Ethn Dis. 2003;13:186 –192.
14
Circulation
August 28, 2007
Downloaded from http://circ.ahajournals.org/ by guest on June 17, 2017
30. Goodwill KE, Sabatier C, Marks C, Raag R, Fitzpatrick PF, Stevens RC.
Crystal structure of tyrosine hydroxylase at 2.3 A and its implications for
inherited neurodegenerative diseases. Nat Struct Biol. 1997;4:578 –585.
31. Mendelsohn ME, Karas RH. Molecular and cellular basis of cardiovascular gender differences. Science. 2005;308:1583–1587.
32. Oparil S, Wright JT Jr. Ethnicity and blood pressure. J Clin Hypertens
(Greenwich). 2005;7:357–364.
33. Thomas KS, Nelesen RA, Ziegler MG, Bardwell WA, Dimsdale JE. Job
strain, ethnicity, and sympathetic nervous system activity. Hypertension.
2004;44:891– 896.
34. Stein CM, Lang CC, Singh I, He HB, Wood AJ. Increased vascular
adrenergic vasoconstriction and decreased vasodilation in blacks: additive
mechanisms leading to enhanced vascular reactivity. Hypertension. 2000;
36:945–951.
35. Boomsma D, Busjahn A, Peltonen L. Classical twin studies and beyond.
Nat Rev Genet. 2002;3:872– 882.
36. MacGregor AJ, Snieder H, Schork NJ, Spector TD. Twins: novel uses to
study complex traits and genetic diseases. Trends Genet. 2000;16:
131–134.
37. Altshuler D, Brooks LD, Chakravarti A, Collins FS, Daly MJ, Donnelly
P. A haplotype map of the human genome. Nature. 2005;437:1299 –1320.
38. Terwilliger JD, Hiekkalinna T. An utter refutation of the “Fundamental
Theorem of the HapMap”. Eur J Hum Genet. 2006;14:426 – 437.
39. O’Connor DT, Insel PA, Ziegler MG, Hook VY, Smith DW, Hamilton
BA, Taylor PW, Parmer RJ. Heredity and the autonomic nervous system
in human hypertension. Curr Hypertens Rep. 2000;2:16 –22.
40. Lillie EO, O’Connor DT. Early phenotypic changes in hypertension: a
role for the autonomic nervous system and heredity. Hypertension. 2006;
47:331–333.
41. Kraft P, Hunter D. Integrating epidemiology and genetic association: the
challenge of gene-environment interaction. Philos Trans R Soc Lond B
Biol Sci. 2005;360:1609 –1616.
42. Imumorin IG, Dong Y, Zhu H, Poole JC, Harshfield GA, Treiber FA,
Snieder H. A gene-environment interaction model of stress-induced
hypertension. Cardiovasc Toxicol. 2005;5:109 –132.
43. Kennedy BP, Rao F, Botiglieri T, Sharma S, Lillie EO, Ziegler MG,
O’Connor DT. Contributions of the sympathetic nervous system, glutathione, body mass and gender to blood pressure increase with normal
aging: influence of heredity. J Hum Hypertens. 2005;19:951–969.
44. King D, Etzel JP, Chopra S, Smith J, Cadman PE, Rao F, Funk SD, Rana
BK, Schork NJ, Insel PA, O’Connor DT. Human response to alpha2adrenergic agonist stimulation studied in an isolated vascular bed in vivo:
biphasic influence of dose, age, gender, and receptor genotype. Clin
Pharmacol Ther. 2005;77:388 – 403.
45. Zaitlen NA, Kang HM, Feolo ML, Sherry ST, Halperin E, Eskin E.
Inference and analysis of haplotypes from combined genotyping studies
deposited in dbSNP. Genome Res. 2005;15:1594 –1600.
46. Zaykin DV, Westfall PH, Young SS, Karnoub MA, Wagner MJ, Ehm
MG. Testing association of statistically inferred haplotypes with discrete
and continuous traits in samples of unrelated individuals. Hum Hered.
2002;53:79 –91.
47. Stram DO, Leigh Pearce C, Bretsky P, Freedman M, Hirschhorn JN,
Altshuler D, Kolonel LN, Henderson BE, Thomas DC. Modeling and
E-M estimation of haplotype-specific relative risks from genotype data
for a case-control study of unrelated individuals. Hum Hered. 2003;55:
179 –190.
48. Knowler WC, Williams RC, Pettitt DJ, Steinberg AG. Gm3;5,13,14 and
type 2 diabetes mellitus: an association in American Indians with genetic
admixture. Am J Hum Genet. 1988;43:520 –526.
CLINICAL PERSPECTIVE
Tyrosine hydroxylase (TH) is the rate-limiting enzyme in catecholamine biosynthesis. We undertook systematic
polymorphism discovery at the TH locus and then tested variants for contributions to sympathetic function and blood
pressure, initially in twin pairs to probe heritability and then in the population. We found that catecholamine secretory traits
are heritable, displaying joint genetic determination with autonomic activity and finally with blood pressure in the
population. Interindividual differences in catecholamine secretion are influenced by genetic variation in the adrenergic
pathway encoding catecholamine synthesis, especially at the classically rate-limiting step, TH. These results document
novel pathophysiological links between a key adrenergic locus, catecholamine metabolism, and blood pressure and suggest
new strategies to approach the mechanism, diagnosis, and treatment of systemic hypertension.
Downloaded from http://circ.ahajournals.org/ by guest on June 17, 2017
Tyrosine Hydroxylase, the Rate-Limiting Enzyme in Catecholamine Biosynthesis.
Discovery of Common Human Genetic Variants Governing Transcription, Autonomic
Activity, and Blood Pressure In Vivo
Fangwen Rao, Lian Zhang, Jennifer Wessel, Kuixing Zhang, Gen Wen, Brian P. Kennedy,
Brinda K. Rana, Madhusudan Das, Juan L. Rodriguez-Flores, Douglas W. Smith, Peter E.
Cadman, Rany M. Salem, Sushil K. Mahata, Nicholas J. Schork, Laurent Taupenot, Michael G.
Ziegler and Daniel T. O'Connor
Circulation. published online August 13, 2007;
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Copyright © 2007 American Heart Association, Inc. All rights reserved.
Print ISSN: 0009-7322. Online ISSN: 1524-4539
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ON-LINE MATERIALS AND METHODS SUPPLEMENT.
Subjects and clinical characterization. Initially, a series of n=80 unrelated,
ethnically diverse individuals was studied by resequencing of TH for systematic
polymorphism discovery, with individuals selected to span a diverse range of
biogeographic ancestries for initial polymorphism discovery: n=23 European
ancestry (white), n=25 sub-Saharan African ancestry (black), n=16 east-Asian
ancestry (Asian), and n=16 Mexican-American ancestry (Hispanic). A series of
n=213 twin pairs (of several ancestries) was later resequenced at the TH
promoter.
Ethnicity was established by self-identification, as well as that of the parents
and grandparents. None of the subjects had a history of renal failure. Definitions
of subject characteristics are according to previous reports from our laboratory. 1
Subjects were volunteers from southern California, and each subject gave
informed, written consent; the protocol was approved by the institutional review
board.
Twin pairs. We recruited a series of twin pairs, taking advantage of a large
population-based twin registry in southern California 2,3 as well as by
advertisement 2. These twin individuals were all of European ancestry, to permit
allelic association studies within one ethnicity. There were n=344 individuals from
n=172 Caucasian (European ancestry) twin pairs; n=119 MZ pairs (24 M/M, 95
F/F); and 53 DZ pairs (9 M/M, 33 F/F, 11 M/F). The twin ages were 15 to 84
years old. 34 of the 344 twin subjects had essential hypertension (9 male, 25
female); 30 were treated for hypertension. Family history of hypertension was:
75/172 twin pairs (44%) were family history positive for hypertension (one or both
parents), while 83/172 pairs (56%) were negative. Twin zygosity (MZ or DZ) was
confirmed by use of either >100 microsatellites (chromosomes 1 and 2) for selfidentified DZ twins, or SNP data (11-177 SNPs) as well as the TH (TCAT)n
microsatellite 2 for self-reported MZ twins. In previous studies of the intronic
(TCAT)n polymorphism 2, we reported on catecholamine secretion in a smaller,
earlier subset (n=178 individuals) of the current twin sample.
Hypertension and population blood pressure extremes.
Subjects: Subjects were sampled from 53,078 individuals (27,475 females
and 25,538 males) whose medical information, as well as genomic DNA, were
obtained through routine, yearly health appraisal visits to Kaiser-Permanente
medical group, a subscription-based, primary care, health maintenance
organization located in San Diego, CA. 81% of enrollees attended this health
appraisal clinic, while 46% of eligible subjects gave informed consent, and were
therefore considered for enrollment; consenting subjects were slightly older
(58±14 versus 51±16 years) and more likely to be men (50 versus 45%) than
non-consenting subjects, but did not differ in frequency of self-reported
cardiovascular disease. Blood pressure was measured in seated subjects using
aneroid sphygmomanometry. If DBP was elevated, repeat measurement was
obtained. Blood for preparation of genomic DNA was obtained with informed
consent, and samples were de-identified.
Selection criteria: Males and females were selected from the highest and
lowest (extreme) percentiles of DBP distribution. Subjects were ascertained by
using DBP as the trait because twin and family studies provide evidence that
DBP is substantially heritable 4-6. Ethnicity, defined by self-identification, including
that of both parents and all four grandparents, was specified as white (European)
ancestry. Subjects in the upper DBP percentiles were diagnosed with
hypertension based on repeated blood pressure measurements and did not have
renal failure (serum creatinine concentration was ≤1.5 mg/dl in 98.6% of
subjects). Ages of subjects at the lower extreme did not significantly differ from
those selected from the upper extreme. Since males and females have different
BP distributions in the population, separate selections were done for men and
women. Individuals with higher DBPs (≥92 mmHg) had values in the upper 4.9th
percentile of the overall DBP distributions, while individuals with lower DBP
values (<61 mmHg) represented the lower 4.8th percentile; 53% were women.
Elevated DBP values were verified by repeated blood pressure measurements;
48% of the hypertensive subjects reported being prescribed and taking one or
more antihypertensive drugs. Subjects in the lower DBP group did not have
histories of hypertension, nor antihypertensive drugs.
Hypertension replication study. To replicate the effects of TH
polymorphisms on hypertension, we studied 927 subjects from the UCSD and
San Diego V.A. populations. Among subjects with a diagnosis of essential
hypertension, 58% were treated with antihypertensive drugs; the average blood
pressures were 149±1/84±1 mmHg. In the subjects with normal blood pressure,
the average blood pressures were 122±1/70±1 mmHg (none were on
antihypertensive medications). 24% of subjects were women. Characterization of
hypertensive subjects has been reported previously 1.
Genomics. Genomic DNA was prepared from leukocytes in EDTAanticoagulated blood, using PureGene extraction columns (Gentra Biosystems,
Minnesota) as described 2. Public draft human and mouse genome sequence
was obtained from the UCSC Genome Bioinformatics website and used as a
scaffold for primer design and sequence alignment. The base position numbers
were from NCBI TH (isoform b) source clones NM_000360, NT_009237.17, and
NP_000351. Promoter positions were numbered with respect to (-) the TH open
reading frame start codon (ATG). PCR primers were designed by Primer3 7 to
span -1155 bp of the proximal promoter, and each of the 13 exons with 50-100
bp of flanking intron. Target sequences were amplified by PCR from 20 ng
genomic DNA in a final volume of 25 µl, which also contained 0.1 unit of Taq
DNA polymerase (Applied Biosystems), 200 µM of each dNTP, 300 nM of each
primer, 50 mM KCl, and 2 mM MgCl2. PCR was performed in a MJ PTC-225
thermal cycler, starting with 12 minutes of denaturation at 95°C, followed by 45
cycles at 95°C for 30 sec, 63°C for 1 minute (annealing), and 72°C (extension)
for 1 minute, and then a final extension of 8 minutes at 72°C. PCR products were
treated with exonuclease I and shrimp alkaline phosphatase to remove primers,
and then dNTPs prior to cycle sequencing with BigDye terminators (Applied
Biosystems). Sequence was determined on an ABI 3100 automated sequencer,
and analyzed using the Phred/Phrap/Consed suite of software to provide base
quality scores, detecting polymorphism and heterozygosity using PolyPhred 8
and manually confirmed. A subset of the data was cross-validated manually
using base calls from Applied Biosystems software and visual inspection of trace
files to identify heterozygotes. Rare SNPs were confirmed by re-sequencing in
multiple individuals, and from the reverse direction. In addition to initial SNP
discovery in a panel of 80 genomic DNA samples, the proximal promoter region
of TH was resequenced for SNPs scoring in all 172 twin pairs.
Comparative genomics. The same TH promoter region was also
resequenced in three non-human primates, with genomic DNA obtained from the
Coriell Institute (Camden, NJ): one chimpanzee (NA03448A), one gorilla
(NG05251B), and one orangutan (NG12256).
Single nucleotide polymorphism. The Val81Met polymorphism in tyrosine
hydroxylase exon 2 (rs6356, A/G) was scored in a two-stage assay9. In stage
one, PCR primers flanking the polymorphism amplified the target region from 5
ng of genomic DNA. In stage two, an oligonucleotide primer flanking the variant
was annealed to the amplified template, and extended across the variant base.
The mass of the extension product (wild-type versus variant) was scored by
MALDI-TOF mass spectrometry (low mass allele versus high mass allele). In
n=440 individuals ascertained from twin families, genotypic ratios were: A/A, 72;
A/G, 197; G/G, 171. Allele frequencies were: Met=A=39%, Val=G=61%; HardyWeinberg equilibrium χ2=1.41, p=0.234.
Biochemical phenotyping in twin pairs: Catecholamines.
Plasma and urine catecholamines were measured radioenzymatically 10. The
assay uses a pre-concentration step that increases sensitivity by ~10-fold over
other COMT-based assays, and ~20-fold over many HPLC assays, permitting
accurate measurement of basal plasma epinephrine levels, which are at the limit
of sensitivity for HPLC assays. Urine catecholamine values were normalized to
creatinine excretion in the same sample.
Physiological/autonomic phenotyping in twin pairs in vivo.
Prolonged recording of blood pressure and heart rate. Blood pressure (in
mmHg) and pulse interval (R-R interval or heart period, in msec/beat) were
recorded continuously and non-invasively for 5 minutes in seated subjects with a
radial artery applanation device and dedicated sensor hardware (Colin Pilot;
Colin Instruments, San Antonio, TX) and software (ATLAS, WR Medical,
Stillwater, MN; and ANS-TDA [Autonomic Nervous System, Tonometric Data
Analysis], Colin Instruments, San Antonio, TX), calibrated every 5 minutes
against ipsilateral brachial arterial pressure with a cuff sphygmomanometer 2.
Heart rate was recorded continuously with thoracic EKG electrodes to the Colin
Pilot. Average, maximum, and minimum values, as well as coefficients of
variation were calculated for blood pressure and pulse interval, using the ANSTDA software.
Environmental stress: the cold pressor test (CPT). BP and HR were
recorded continuously and non-invasively with a calibrated radial artery
applanation device and dedicated sensor hardware (Colin Pilot; Colin
Instruments, San Antonio, TX) and software (ATLAS, WR Medical, Stillwater,
MN; TDA [Tonometric Data Analysis], Colin Instruments, San Antonio, TX) during
the CPT (immersion of the left hand in ice water for 60 seconds, after a 10
minute rest 2,11,12. We identified at least 3 beats with stable (within ±10%) values
for BP and HR just before and at the end of the CPT.
Baroreceptor sensitivity (slope) in the time domain. Blood pressure and
heart rate were also continuously recorded with the same devices during
spontaneous excursions of blood pressure with reciprocal heart rate changes:
upward excursions of blood pressure with reflex bradycardia, and downward
excursions of blood pressure with reflex tachycardia. In each case, baroreceptor
slope in the “time domain” 11,12 was calculated with the ANS-TDA software, with
beat-by-beat regression of change in SBP (SBP, mmHg) as a function of change
in pulse interval (R-R interval; msec/beat) on the succeeding beat (phase lag = 1
beat). Time windows of >4 beats were used, with SBP of >1 mmHg and R-R of
>6 msec. Baroreceptor slope (msec/mmHg) values were recorded for
regressions with target correlation coefficients of r>0.9. The slopes for 3 such
regressions, if each was within ±10% of the mean value, were averaged to yield
the final value for baroreceptor slope. Baroreceptor slopes were separately
determined for upward and downward spontaneous excursions of blood
pressure.
Tyrosine hydroxylase promoter haplotype activity in vitro.
Human TH promoter/reporter plasmids were constructed, sequence-verified,
and studied as previously described for other neuroendocrine promoters 13, 14,15.
Promoter positions were numbered upstream (-) or downstream (+) of the start
codon (A in ATG as +1 bp). Exon 1 of human TH (isoform b) includes a 19-bp 5’UTR (5’-CGGACCTCCACACTGAGCC-3’) just upstream of the ATG start codon.
A haplotype-specific promoter fragment (from a wild-type haplotype
homozygote), corresponding to TH -957 bp/-1 bp, was PCR-amplified from
genomic DNA of known homozygotes; thus, the promoter amplicon included the
9-bp 5’-UTR. The promoter amplicon was subcloned into the promoter-less firefly
luciferase reporter plasmid pGL3-Basic (Promega, Madison, WI). Only the wildtype TH haplotype was amplified from genomic DNA. The other haplotypes were
generated by point mutation of the wild-type promoter haplotype-firefly luciferase
reporter plasmid (QuikChange mutagenesis; Stratagene) and resequenced to
verify the identity of each allele and haplotype. Supercoiled plasmids were
purified on columns (Qiagen, Valencia, CA), prior to transfection. PC12
pheochromocytoma cells were transfected (at 50-60% confluence, 1 day after 1:4
splitting) with 1 µg of supercoiled promoter haplotype-firefly luciferase reporter
plasmid and 10 ng of the Renilla luciferase expression plasmid pRL-TK
(Promega Inc., Madison, WI) as an internal control per well, by the liposome
method (Superfect, Qiagen, Valencia, CA). The firefly and Renilla luciferase
activities in the cell lysates were measured 48 hours after transfection, and the
results were expressed as the ratio of firefly/Renilla luciferase activity (“Stop &
Glo®”, Promega, Madison, WI). Each experiment was repeated six times.
1.
O'Connor DT, Kailasam MT, Kennedy BP, Ziegler MG, Yanaihara N,
Parmer RJ. Early decline in the catecholamine release-inhibitory peptide
catestatin in humans at genetic risk of hypertension. J Hypertens.
2002;20:1335-45.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
Zhang L, Rao F, Wessel J, Kennedy BP, Rana BK, Taupenot L, Lillie EO,
Cockburn M, Schork NJ, Ziegler MG, O'Connor DT. Functional allelic
heterogeneity and pleiotropy of a repeat polymorphism in tyrosine
hydroxylase: prediction of catecholamines and response to stress in twins.
Physiol Genomics. 2004;19:277-91.
Cockburn M, Hamilton A, Zadnick J, Cozen W, Mack TM. The occurrence
of chronic disease and other conditions in a large population-based cohort
of native Californian twins. Twin Res. 2002;5:460-7.
Evans A, Van Baal GC, McCarron P, DeLange M, Soerensen TI, De Geus
EJ, Kyvik K, Pedersen NL, Spector TD, Andrew T, Patterson C, Whitfield
JB, Zhu G, Martin NG, Kaprio J, Boomsma DI. The genetics of coronary
heart disease: the contribution of twin studies. Twin Res. 2003;6:432-41.
Kupper N, Willemsen G, Riese H, Posthuma D, Boomsma DI, de Geus
EJ. Heritability of daytime ambulatory blood pressure in an extended twin
design. Hypertension. 2005;45:80-5.
Snieder H, Harshfield GA, Treiber FA. Heritability of blood pressure and
hemodynamics in African- and European-American youth. Hypertension.
2003;41:1196-201.
Rozen S, Skaletsky H. Primer3 on the WWW for general users and for
biologist programmers. Methods Mol Biol. 2000;132:365-86.
Nickerson DA, Tobe VO, Taylor SL. PolyPhred: automating the detection
and genotyping of single nucleotide substitutions using fluorescencebased resequencing. Nucleic Acids Res. 1997;25:2745-51.
Buetow KH, Edmonson M, MacDonald R, Clifford R, Yip P, Kelley J, Little
DP, Strausberg R, Koester H, Cantor CR, Braun A. High-throughput
development and characterization of a genomewide collection of genebased single nucleotide polymorphism markers by chip-based matrixassisted laser desorption/ionization time-of-flight mass spectrometry. Proc
Natl Acad Sci U S A. 2001;98:581-4.
Kennedy B, Ziegler MG. A more sensitive and specific radioenzymatic
assay for catecholamines. Life Sci. 1990;47:2143-53.
Parmer RJ, Cervenka JH, Stone RA, O'Connor DT. Autonomic function in
hypertension. Are there racial differences? Circulation. 1990;81:1305-11.
O'Connor DT, Insel PA, Ziegler MG, Hook VY, Smith DW, Hamilton BA,
Taylor PW, Parmer RJ. Heredity and the autonomic nervous system in
human hypertension. Curr Hypertens Rep. 2000;2:16-22.
Rozansky DJ, Wu H, Tang K, Parmer RJ, O'Connor DT. Glucocorticoid
activation of chromogranin A gene expression. Identification and
characterization of a novel glucocorticoid response element. J Clin Invest.
1994;94:2357-68.
Wu RA, Kailasam MT, Cervenka JH, Parmer RJ, Kennedy BP, Ziegler
MG, O'Connor DT. Does lipophilicity of angiotensin converting enzyme
inhibitors selectively influence autonomic neural function in human
hypertension? J Hypertens. 1994;12:1243-7.
Wen G, Mahata SK, Cadman P, Mahata M, Ghosh S, Mahapatra NR, Rao
F, Stridsberg M, Smith DW, Mahboubi P, Schork NJ, O'Connor DT,
Hamilton BA. Both rare and common polymorphisms contribute functional
variation at CHGA, a regulator of catecholamine physiology. Am J Hum
Genet. 2004;74:197-207.
12-10-06.
On-line Tables.
Table I. Polymorphism discovery at the human tyrosine hydroxylase (TH) promoter. Results from 2n=586 chromosomes.
Summary of tyrosine hydroxylase (TH) promoter SNP discovery
Minor allele frequency in population
(2n = number of chromosomes)
a
SNP # SNP Contig bp
d
SNP position in promoter
1
c/T
957789
-833
2
c/T
957780
-824
3
g/A
957770
-814
4
g/C
957757
-801
b
Hardy Weinberg equilibrium
RefSNP Asian (2n=60) Black (2n=78) Hispanic (2n=70) White (2n=378) Total (2n=586)
rs10770141
rs10840490
0
0.09
0
0.003
0.167
0.603
0.357
0
0
0
0.1
0.09
0.2
χ
2
P
0.014
0.001
0.977
0.336
0.57
0.136
0.713
0.003
0.002
0.01
0.917
0.12
0.123
0.196
0.658
5
t/C
957697
-741
0
0.013
0
0
0.002
-
NC
6
a/G
957597
-641
0
0.064
0.029
0
0.012
-
NC
7
a/G
957537
-581
rs10770140
0.1
0.59
0.314
0.354
0.355
0.003
0.959
8
g/A
957450
-494
rs11042962
0.717
0.179
0.371
0.484
0.454
0.677
0.411
9
c/T
957344
-388
0
0
0
0.003
0.002
0.01
0.917
10
g/T
957050
-94
0
0.013
0
0
0.002
-
NC
a: Lower case indicates major allele, upper case indicates minor allele (major/Minor)
b: Positions are numbered upstream (-) or downstream (+) of the start codon (ATG)
c: HWE calculation done on one ethnicity (white)
d: Contig NT_028310.
Common (minor allele frequency >10%) SNPs are shown in bold type. NC: not calculable, since these SNPs were monomorphic in the largest (white) population.
c
Table II. Descriptive statistics for the twin study population: n=344 individuals from n=172 Caucasian (European ancestry) twin pairs; n=119
MZ pairs (24 M/M, 95 F/F); and 53 DZ pairs (9 M/M, 33 F/F, 11 M/F). Entries reflect mean value ± SEM, by GEE (generalized estimator
equations). *: significant (p<0.05) differences are given in bold type.
Trait
All
Male
Sex
Female
<40 years
Age
≥40 years
n=77
n=267
p
n=164
n=180
p
24.9±0.26
40.8±1.3
25.6±0.61
40.8±1.3
24.7±0.39
0.0910
0.2204
26.1±0.7
23.5±0.39
54.2±1.1
26.1±0.50
<0.0001*
<0.0001*
118±1.2
63.7±0.9
848±7.7
123±2.8
65.1±2.4
851±21.0
116±1.5
63.3±1.1
848±10.5
0.0438*
0.4918
0.9049
111±1.6
59.4±1.2
843±13.2
124±2.0
67.7±1.5
854±13.7
<0.0001*
<0.0001*
0.5654
117±0.96
63.8±0.6
71.8±0.7
130.0±1.3
74.4±0.8
75.7±0.7
12.5±0.99
10.6±0.6
3.83±0.5
120±2.1
71.3±1.7
71.3±1.7
134±2.9
77.5±1.7
72.8±1.7
12.8±1.6
12.8±1.6
1.5±1.4
116±1.3
71.8±0.9
71.9±0.9
128±1.8
73.5±1.1
76.5±0.9
9.9±0.8
9.9±0.8
4.5±0.7
0.1343
0.8073
0.7877
0.0754
0.0553
0.0654
0.1036
0.1034
0.0540
112±1.2
60.3±0.79
72.2±1.2
121±1.9
71.1±1.2
76.2±1.2
10.7±0.9
10.7±1.0
3.9±1.1
122±1.7
66.9±1.1
71.3±1.1
137±2.2
77.5±1.4
75.2±1.1
10.4±0.9
10.4±0.9
3.77±0.6
<0.0001*
<0.0001*
0.5826
<0.0001*
0.0008*
0.5423
0.8558
0.8546
0.9081
15.0±0.6
12.3±0.5
14.7±1.3
12.5±1.2
15.1±0.8
12.2±0.7
0.7975
0.8492
19.4±1.1
15.7±1.0
10.9±0.6
9.1±0.6
<0.0001*
<0.0001*
21.4±1.3
336±11.4
32.1±4.2
355±26.8
18.6±1.3
330±15.9
0.0065*
0.4262
22.6±1.9
295±14.0
20.2±2.2
374±22.7
0.4163
0.0045*
n=344
DEMOGRAPHIC/PHYSICAL
Age, years
Body mass index, kg/m2
40.8±0.9
PHYSIOLOGICAL
Prolonged (5 minute) basal
monitoring
Basal SBP, mmHg
Basal DBP, mmHg
Basal pulse (R-R)
interval, msec/beat
Cold stress
Basal SBP, mmHg
Basal DBP, mmHg
Basal HR, beats/min
Post SBP, mmHg
Post DBP, mmHg
Post HR, beats/min
SBP change, mmHg
DBP change, mmHg
HR change, beats/min
AUTONOMIC
Baroreceptor slope,
msec/mmHg
Upward deflections
Downward deflections
BIOCHEMICAL
Plasma epinephrine, pg/ml
Plasma norepinephrine,
pg/ml
Urinary epinephrine, ng/gm
Urinary norepinephrine,
ng/gm
12113±331
28697±924
9771±691
13526±1337 9879±686 0.0284* 11497±990
32447±3319 23455±1519 0.0192* 23929±2017 26613±2049
0.1575
0.3529
Table III. Trait correlations in twin pairs. Inter-individual correlations among physiological and biochemical variables in the twins. Entries
below the diagonal reflect parametric Pearson product-moment correlations (r); entries below the diagonal reflect non-parametric
Spearman’s rank sum correlations (rho). All significance (p) values are 2-tailed. *: p<0.05, given in bold type. ∆SBP: change in SBP during
the cold pressor test. ∆DBP: change in DBP during the cold pressor test. ∆HR: change in HR during the cold pressor test. PRESBP: SBP
prior to cold pressor test. PREDBP: DBP prior to cold pressor test. PREHR: heart rate prior to cold pressor test. POSTSBP: SBP at the
end of the cold pressor test. POSTDBP: DBP at the end of the cold pressor test. POSTHR: HR at the end of the cold pressor test. BLIPUP
SLOPE: Baroreceptor slope, upward deflections. BLIPDOWN SLOPE: Baroreceptor slope, downward deflections. pE: plasma epinephrine.
pNE: plasma norepinephrine. uE: urinary epinephrine excretion. uNE: urinary norepinephrine excretion. Results are from one twin per
twinship.
Correlations: Non-parametric (Spearman rho)
Parametric
(Pearson r).
Correlation
∆DBP
∆DBP
∆HR
∆SBP
PREHR
PRE
SBP
POST
PRE DBP DBP
POST
HR
POST BLIPUP BLIPDOWN
SBP SLOPE SLOPE
pE
pNE
uE
uNE
1.000
0.201
0.708
-0.045
-0.048
-0.129
0.616
0.131
0.436
-0.096
0.066
-0.069
0.018
0.005
-0.049
171
0.008
171
0.000
171
0.557
171
0.533
171
0.094
170
0.000
171
0.087
171
0.000
171
0.221
165
0.401
164
0.381
162
0.820
162
0.949
157
0.545
157
0.140
1.000
0.085
-0.347
-0.013
-0.058
0.097
0.424
0.038
0.059
0.153
-0.173
-0.005
-0.117
-0.079
171
0.271
171
0.000
171
0.866
171
0.456
170
0.208
171
0.000
171
0.621
171
0.455
165
0.050
164
0.028
162
0.954
162
0.146
157
0.327
157
0.714
0.015
1.000
-0.011
0.126
0.119
0.618
0.075
0.714
-0.237
-0.016
-0.136
0.126
-0.008
0.078
0.845 .
171
171
0.882
171
0.101
171
0.122
170
0.000
171
0.332
171
0.000
171
0.002
165
0.838
164
0.083
162
0.111
162
0.923
157
0.329
157
1.000
0.134
0.124
0.057
0.651
0.083
-0.308
-0.331
0.129
-0.016
0.072
0.123
171
0.080
171
0.108
170
0.459
171
0.000
171
0.278
171
0.000
165
0.000
164
0.101
162
0.838
162
0.369
157
0.126
157
1.000
0.645
0.484
0.107
0.726
-0.224
-0.284
0.089
0.114
0.036
0.312
171
0.000
170
0.000
171
0.163
171
0.000
171
0.004
165
0.000
164
0.260
162
0.147
162
0.653
157
0.000
157
1.000
0.650
0.015
0.516
-0.234
-0.309
0.085
0.038
-0.004
0.242
170
0.000
170
0.849
170
0.000
170
0.003
164
0.000
163
0.284
161
0.630
161
0.957
156
0.002
156
1.000
0.107
0.757
-0.265
-0.193
-0.008
0.045
-0.004
0.133
171
0.165
171
0.000
171
0.001
165
0.013
164
0.915
162
0.566
162
0.958
157
0.096
157
1.000
0.119
-0.288
-0.227
-0.030
0.007
-0.062
0.067
0.123
0.000
0.003
0.700
0.925
0.440
0.402
∆HR
p
N
Correlation
∆SBP
p
N
Correlation
0.000
171
PREHR
p
N
Correlation
0.012
-0.382
0.054
0.879
171
0.000
171
0.483 .
171
PRESBP
p
N
Correlation
-0.056
-0.029
0.070
0.156
0.466
171
0.702
171
0.361
171
0.041 .
171
PREDBP
p
N
Correlation
-0.121
-0.065
0.101
0.131
0.628
0.117
170
0.398
170
0.191
170
0.089
170
0.000 .
170
POSTDBP
p
N
Correlation
0.648
0.051
0.603
0.112
0.441
0.675
0.000
171
0.509
171
0.000
171
0.146
171
0.000
171
0.000 .
170
POSTHR
p
N
Correlation
0.119
0.425
0.061
0.673
0.130
0.076
0.147
p
0.122
0.000
0.425
0.000
0.091
0.322
0.056 .
.
0.068 .
171
p. 2
N
Correlation
171
171
171
171
171
170
171
171
171
165
164
162
162
157
157
0.490
0.014
0.752
0.122
0.650
0.527
0.766
0.128
1.000
-0.332
-0.221
-0.016
0.168
0.059
0.274
0.000
171
0.855
171
0.000
171
0.113
171
0.000
171
0.000
170
0.000
171
0.096 .
171
171
0.000
165
0.005
164
0.842
162
0.033
162
0.465
157
0.001
157
-0.111
0.051
-0.243
-0.371
-0.274
-0.325
1.000
0.690
-0.179
-0.243
-0.050
-0.293
p
N
Correlation
0.158
165
0.513
165
0.002
165
0.000
165
0.000
165
166
0.000
165
0.025
157
0.002
157
0.541
152
0.000
152
0.003
0.125
-0.068
-0.351
-0.292
1.000
-0.260
-0.150
-0.046
-0.279
0.971
164
0.110
164
0.387
164
0.000
164
0.000
164
0.000
163
165
0.001
156
0.062
156
0.575
151
0.001
151
pE
p
N
Correlation
-0.074
-0.195
-0.130
0.127
-0.168
1.000
0.228
0.337
0.082
0.346
162
0.013
162
0.098
162
163
0.003
163
0.000
154
0.311
154
pNE
p
N
Correlation
0.109
-0.003
1.000
0.158
0.459
0.168
162
163
0.051
154
0.000
154
uE
p
N
Correlation
1.000
0.450
158
0.000
158
uNE
1
POSTSBP
BLIPUP
SLOPE
BLIPDOWN
SLOPE
p
N
Correlation
-0.270 -0.289
0.000
165
0.000
165
-0.306 -0.230
-0.242
-0.227
0.709
0.003
164
0.002
164
0.003
164
0.000 .
165
0.003
0.037 -0.036
-0.029
-0.112
-0.075
0.106
162
0.971
162
0.639
161
0.653
162
0.715
162
0.155
162
0.349
157
0.211
0.061
0.129
0.099
0.155
0.065
0.222
-0.272
-0.214
0.206
0.969
162
0.007
162
0.440
162
0.101
162
0.213
161
0.049
162
0.411
162
0.004
162
0.001
157
0.007
156
0.008 .
163
-0.013
-0.120
0.014
0.114
-0.010
-0.011 -0.020
0.009
-0.005
-0.090
-0.097
0.364
0.101
p
N
Correlation
0.871
157
0.134
157
0.862
157
0.154
157
0.899
157
0.895
156
0.802
157
0.909
157
0.946
157
0.270
152
0.235
151
0.000
154
0.215 .
154
0.039
-0.032
0.174
0.102
0.253
0.205
0.181
0.072
0.273
-0.262
-0.237
0.053
0.563
0.424
p
N
0.628
157
0.693
157
0.029
0.203
157
0.001
0.010
0.023
0.001
0.001
0.003
0.000 .
156
157
157
152
151
0.511
154
0.000
157
0.369
157
157
p. 3
0.000
164
-0.318
0.000 .
165
0.036 .
156
154
158
158
Table IV. Heritability (h2=VG/VP) of autonomic function in twin pairs: biochemical and physiological traits. Heritability (± SEM) is the
percentage of phenotypic variation (VP) explained by additive genetic factors (VG). Values are age- and sex-adjusted. P value is the
significance of the heritability value. *: Significant (p<0.05) values are indicated in bold type. Heritability was determined in SOLAR:
Sequential Oligogenic Linkage Analysis Routines.
Phenotype
DEMOGRAPHIC/PHYSICAL
Weight, kg
Height, meters
PHYSIOLOGICAL
Prolonged (5 minute) basal monitoring
Basal SBP, mmHg
Basal DBP, mmHg
Basal pulse (R-R) interval, msec/beat
Cold stress
Basal SBP, mmHg
Basal DBP, mmHg
Basal HR, beats/min
Post SBP, mmHg
Post DBP, mmHg
Post HR, beats/min
∆SBP, mmHg
∆DBP, mmHg
∆HR, beats/min
AUTONOMIC
Baroreceptor slope, msec/mmHg
Upward deflections
Downward deflection
BIOCHEMICAL
Plasma norepinephrine, pg/ml
Plasma epinephrine, pg/ml
Urinary norepinephrine, ng/gm
Urinary norepinephrine, ng/gm (log)
Urinary epinephrine, ng/gm
p. 4
Heritability
(h2),
% ± SEM
p value
for h2
n (individuals)
87±2
93±1
<0.0001*
<0.0001*
326
326
26±8
18±9
61±6
0.0016*
0.0359*
<0.0001*
326
326
326
30±8
27±9
54±7
29±8
37±8
52±6
23±9
32±8
36±8
0.0002*
0.0018*
<0.0001*
0.0005*
<0.0001*
<0.0001*
0.0098*
0.0003*
<0.0001*
326
326
326
326
326
326
326
326
326
33.3±9.2
43.0±7.3
0.0004*
<0.0001*
329
329
69.9±4.4
66.7±5.9
46.1±6.5
49.6±6.7
67.6±4.9
<0.0001*
<0.0001*
0.0001*
0.0001*
<0.0001*
327
326
316
316
316
Promoter
haplotype
number
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
Chimp
Gorilla
Orangutan
Nucleotide at bp position (upstream of ATG):
-833
C
C
C
C
C
C
T
C
C
C
C
C
C
C
C
C
C
C
T
C
-824
C
T
C
C
T
C
T
C
C
T
T
T
T
T
C
T
T
C
C
C
-814
G
G
G
G
G
A
G
G
G
G
G
G
G
G
G
G
G
G
G
G
-801
G
G
C
G
G
G
G
G
G
G
G
G
G
C
C
G
G
G
G
G
-741
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
C
T
C
C
C
Frequency (2n=no. of chromosomes) in that population:
Asian
Black
Hispanic
White
Total
-641 -581 -494 -388 -94
(2n=60)
(2n=78)
(2n=70)
(2n=378)
(2n=586)
A
A
A
C
G 0.617 (37)
0.154 (12)
0.371 (26)
0.468 (177)
0.430 (252)
A
G
G
C
G 0.100 (6)
0.346 (27)
0.300 (21)
0.323 (122)
0.300 (176)
A
A
G
C
G 0.117 (7)
0.077 (6)
0.171 (12)
0.116 (44)
0.118 (69)
A
C
G 0.000 (0)
0.000 (0)
0.000 (0)
0.008 (3)
0.005 (3)
G
A
A
T
G 0.000 (0)
0.000 (0)
0.000 (0)
0.003 (1)
0.002 (1)
G
G
A
C
G 0.000 (0)
0.000 (0)
0.000 (0)
0.003 (1)
0.002 (1)
A
A
A
C
G 0.000 (0)
0.090 (7)
0.000 (0)
0.003 (1)
0.014 (8)
G
G
A
G
G
C
G 0.017 (1)
0.090 (7)
0.000 (0)
0.019 (7)
0.026 (15)
A
A
G
C
G 0.067 (4)
0.064 (5)
0.086 (6)
0.042 (16)
0.053 (31)
G
C
G 0.000 (0)
0.026 (2)
0.029 (2)
0.000 (0)
0.007 (4)
A
G
A
C
G 0.000 (0)
0.064 (5)
0.029 (2)
0.008 (3)
0.017 (10)
A
G
G
C
G 0.000 (0)
0.038 (3)
0.000 (0)
0.000 (0)
0.005 (3)
G
G
A
C
G 0.083 (5)
0.013 (1)
0.014 (1)
0.005 (2)
0.015 (9)
A
A
A
C
G 0.000 (0)
0.000 (0)
0.000 (0)
0.003 (1)
0.002 (1)
A
G
A
C
G 0.000 (0)
0.013 (1)
0.000 (0)
0.000 (0)
0.002 (1)
A
A
A
C
G 0.000 (0)
0.013 (1)
0.000 (0)
0.000 (0)
0.002 (1)
G
G
A
C
T 0.000 (0)
0.013 (1)
0.000 (0)
0.000 (0)
0.002 (1)
G
G
A
C
G
G
G
A
C
G
G
G
A
C
G
G
G
Table V. Haplotype distribution in the tyrosine hydroxylase (TH) promoter region among four human populations. TH promoter
haplotypes were imputed by PHASE using all SNPs (2n=586 chromosomes/n=293 individuals). Data are taken from SNP discovery by
resequencing unrelated individuals of four ethnicities (east Asian, 2n=60 chromosomes; black, 2n=78; Hispanic, 2n=70; white, 2n=378).
The 4 most common (minor allele frequency >10%) SNPs (C-824T, G-801C, A-581G, G-494A), and five most frequent haplotypes
(haplotypes 1,2,3,8,9), are indicated in bold type. Haplotype 9 possesses the major (most frequent) human allele at each position (though
it comprises only ~5.3% of human chromosomes). Haplotype 8 matches the non-human primate alleles at the 4 most common SNPs
(CGGG). Promoter bp positions are numbered upstream (-) of the translation initiation codon (ATG). The 3 non-human primates were
homozygous at each position.
p. 5
Table VI. Effects of TH promoter haplotypes on biochemical and physiological traits in the twins. Results are displayed for trait
means (age- and sex-adjusted) based on number of copies/genome of the most common variants: haplotypes 1 (Hap1, CGAA), 2 (Hap2,
TGGG), and 3 (Hap3, CCAG). SOLAR: Sequential Oligogenic Linkage Analysis Routines.
Phenotype
Haplotype
Number of copies (n) of that haplotype per
diploid genome
p
N (total=327
individuals)
PHYSIOLOGICAL
Prolonged (5 min)
basal monitoring
Basal SBP, mmHg
Basal DBP, mmHg
Basal pulse (R-R)
interval, msec/beat
Cold stress
Basal SBP, mmHg
Basal DBP, mmHg
Basal HR,
beats/min
Post SBP, mmHg
Post DBP, mmHg
SOLAR
% variation
explained
Hap1
Hap2
Hap3
0
(83)
(131)
(268)
1
(165)
(164)
(55)
2
(79)
(32)
(4)
-
-
Hap1
Hap2
Hap3
Hap1
Hap2
Hap3
Hap1
Hap2
Hap3
120±2.2
166±2.1
118±1.4
64.4±1.6
63.8±1.5
63.5±1.1
857±17.0
963±14.2
848±10.7
117±1.7
118±1.5
118±2.5
63.2±1.4
64.0±1.3
65.7±1.9
837±11.8
836±12.9
877±19.3
117±2.7
121±3.9
110±13.4
64.3±2.0
62.4±2.7
57.4±9.7
859±20.4
843±26.0
934±59.8
0.7736
0.8725
0.9912
0.3997
0.4841
0.1039
0.8160
0.2429
0.7889
-
Hap1
Hap2
Hap3
Hap1
Hap2
Hap3
Hap1
Hap2
Hap3
Hap1
Hap2
Hap3
Hap1
Hap2
Hap3
119±2.1
119±1.6
118±1.3
63.7±1.2
65.0±1.0
64.2±0.8
63.7±1.2
70.9±1.3
72.8±1.1
135±3.0
128±2.4
132±1.9
76.2±1.5
73.3±1.6
75.4±1.2
119±1.6
119±1.6
121±2.6
64.6±1.1
64.2±1.1
66.2±1.5
64.6±1.1
72.6±1.3
67.4±1.7
132±2.1
135±2.1
134±3.7
75.3±1.5
76.4±1.4
76.5±2.1
119±1.8
116±2.9
107±8.2
64.7±1.4
63.0±1.9
53.6±3.4
64.7±1.4
71.3±2.8
63.1±3.8
130±3.1
132±4.6
114±2.9
74.5±2.1
78.0±2.5
58.6±3.1
0.7398
0.7219
0.9113
0.8616
0.3739
0.8720
0.2858
0.5347
0.0059*
0.9361
0.0901
0.8127
0.8375
0.0661
0.5841
3.78%
-
p. 6
Post HR,
beats/min
∆SBP, mmHg
∆DBP, mmHg
∆HR, beats/min
AUTONOMIC
Baroreceptor
slope,
msec/mmHg
Upward
deflections
Downward
deflections
BIOCHEMICAL
Plasma
epinephrine, pg/ml
Plasma
norepinephrine,
pg/ml
Urinary
epinephrine,
ng/gm
Urinary
norepinephrine,
ng/gm
Hap1
Hap2
Hap3
Hap1
Hap2
Hap3
Hap1
Hap2
Hap3
Hap1
Hap2
Hap3
72.6±1.4
73.9±1.4
75.3±1.0
18.0±2.6
10.7±1.8
14.1±1.7
12.6±1.3
8.2±1.2
11.1±0.95
2.50±1.1
2.86±0.98
2.51±0.78
74.4±1.3
74.2±1.3
71.3±1.9
13.1±1.8
16.3±2.2
14.4±2.4
10.7±1.2
12.4±1.1
10.3±1.4
1.9±0.9
1.6±0.98
3.3±1.2
76.0±1.8
77.2±1.8
59.8±2.2
11.9±2.2
16.9±3.1
6.76±5.3
9.72±1.4
14.9±2.1
5.42±5.97
3.93±1.3
5.52±2.1
-3.3±2.5
0.6638
0.3185
0.0104*
0.7723
0.0154*
0.6926
0.6993
0.0004*
0.4149
0.5233
0.6877
0.9617
3.14%
1.90%
3.73%
-
Hap1
Hap2
Hap3
14.4±1.1
15.2±1.2
14.5±0.7
15.5±1.0
15.2±0.8
17.8±1.8
14.6±1.4
12.8±1.7
9.7±3.6
0.8619
0.4972
0.2347
-
Hap1
Hap2
Hap3
12.7±1.1
11.8±0.8
11.7±0.6
12.5±0.8
12.8±0.9
15.4±1.7
11.4±1.1
11.8±1.1
8.6±2.4
0.4891
0.7018
0.0934
-
Hap1
Hap2
Hap3
Hap1
Hap2
Hap3
Hap1
Hap2
Hap3
Hap1
22.2±1.9
18.6±1.7
21.4±1.6
323±21.8
331±21.7
340±14.7
13447±733
11107±504
12304±450
21.5±2.0
22.4±2.0
21.3±2.0
333±17.5
337±18.0
321±25.7
11804±503
12202±454
11590±538
19.6±2.8
23.8±2.8
366±34.8
343±26.2
11003±812
14505±1136
-
0.8370
0.1109
0.8738
0.9152
0.7682
0.5104
0.3280
0.0044*
0.3999
5.70%
-
29435±1356
26372±1207
28919±1480
29070±1342
26707±1430
27125±1535
33280±1888
-
0.8171
0.0125*
0.0592
4.06%
-
Hap2
Hap3
30105±992
p. 7
Table VII (on-line): Effect of TH promoter diploid haplotype (“diplotype”) on autonomic phenotypes. SOLAR: Sequential Oligogenic
Linkage Analysis Routines.
Table VIIa. TH diplotypes resulting from the most common alleles are shown: Haplotype 1 (Hap1) and Haplotype 2 (Hap2).
Tyrosine hydroxylase diploid haplotype (“diplotype”)
Phenotype
N
PHYSIOLOGICAL
Prolonged (5 min)
basal monitoring
Basal SBP, mmHg
Basal DBP, mmHg
Basal pulse (R-R)
interval, msec/beat
Cold stress
Basal SBP, mmHg
Basal DBP, mmHg
Basal HR, beats/min
Post SBP, mmHg
Post DBP, mmHg
Post HR, beats/min
∆SBP, mmHg
∆DBP, mmHg
∆HR, beats/min
SOLAR
Hap1/Hap1
homozygotes
Hap1/Hap2
heterozygotes
Hap2/Hap2
homozygotes
79
121
32
117±2.7
64.5±2.0
118±1.8
63.3±1.6
857±20.9
829±14.0
p
% variation
explained
121±3.9
62.4±2.7
0.9374
0.4067
-
842±25.2
0.8194
-
119±1.9
64.8±1.4
71.5±1.7
130±3.1
74.5±2.1
75.5±1.8
11.4±2.2
9.7±1.5
3.81±1.4
119±2.0
64.1±1.3
73.0±1.5
133±2.4
76.2±1.7
74.8±1.4
13.9±2.2
12.1±1.3
1.81±1.2
117±3.1
63.1±1.9
71.0±2.8
133±4.4
78.5±2.4
76.8±1.6
16.6±3.2
15.1±2.0
5.51±2.1
0.5572
0.9644
0.3742
0.5373
0.8792
0.5868
0.1604
0.0312*
0.8549
3.47%
-
14.5±1.5
11.4±1.1
15.0±1.0
12.4±1.0
12.7±1.7
11.7±1.0
0.3221
0.5491
-
19.6±2.7
22.7±2.5
24.3±2.8
0.2010
-
367±35.4
348±21.6
351±26.1
0.4623
-
10947±797
11925±574
14383±1175
0.6381
-
27179±1653
29894±1724
33330±1927
0.8624
-
Baroreceptor
slope,
msec/mmHg
Upward deflections
Downward deflections
Biochemical
Plasma epinephrine,
pg/ml
Plasma
norepinephrine, pg/ml
Urinary epinephrine,
ng/gm
Urinary
norepinephrine,
p. 8
ng/gm
p. 9
Table VIIb (on-line). Diploid haplotypes: Haplotypes 1 (Hap1) and 3 (Hap3).
Phenotype
N
PHYSIOLOGICAL
Prolonged (5 min) monitoring
Basal SBP, mmHg
Basal DBP, mmHg
Basal pulse (R-R) interval, msec/beat
Cold stress
Basal SBP, mmHg
Basal DBP, mmHg
Basal HR, beats/min
Post SBP, mmHg
Post DBP, mmHg
Post HR, beats/min
SBP, mmHg
DBP, mmHg
HR, beats/min
AUTONOMIC
Baroreceptor slope, msec/mmHg
Upward deflections
Downward deflections
BIOCHEMICAL
Plasma epinephrine, pg/ml
Plasma norepinephrine, pg/ml
Urinary epinephrine, ng/gm
Urinary norepinephrine, ng/gm
Tyrosine hydroxylase diploid haplotype (haplotypes 1
& 3) effects on traits (mean ± SEM, by GEE)
Hap1/Hap1
Hap1/Hap3 heterozygotes,
or Hap3/Hap3 homozygotes
homozygotes
SOLAR
P
% variation
explained
79
24
118±2.7
65.9±2.4
844±27.4
117±4.3
65.0±2.7
894±26.2
0.7391
0.3131
0.7422
-
117±1.6
64.9±1.3
72.6±1.6
128±3.3
74.4±2.3
76.1±2.0
11.2±2.4
9.34±1.6
3.25±1.5
116±3.5
66.5±2.3
65.7±2.5
128±5.6
74.7±3.6
72.4±2.5
11.3±3.4
8.10±2.4
5.89±1.5
0.7094
0.6133
0.0335*
0.8854
0.9818
0.3093
0.9879
0.5946
0.3324
4.13%
-
14.6±1.5
11.4±1.1
21.0±3.3
15.3±1.9
0.0476*
0.0732
4.49%
-
20.7±3.0
344±37.1
9885±767
26758±1706
20.3±2.6
335±32.3
10532±928
25376±2448
0.8216
0.9483
0.6286
0.7757
-
p. 10
Figure 1. Discovery of four common SNPs (with flanking sequences) in the human tyrosine hydroxylase (TH) proximal
promoter. A, C-824T (C/C C/T, T/T); B, G-801C (G/G, G/C, C/C); C, A-581G (A/A, A/G, G/G); D, G-494A (G/G, G/A, A/A).
Red arrows indicate the SNP positions. Results are ABI-3100 sequence tracings analyzed in PolyPhred.
G-801C
G-494A
A-581G
C-824T
Discovery of 4 common SNPs in the human
tyrosine hydroxylase (TH) proximal promoter
Online I
Pleiotropy: Tyrosine hydroxylase promoter C-824T genotype
jointly predicts autonomic biochemistry and physiology in twins
24
SOLAR (sex- and age-adjusted):
Urine norepinephrine: p=0.0069*, % variation = 1.52%
∆SBP: p=0.01*, % variation = 1.54%
2
Change in SBP post cold stress, mmHg
Bivariate: χ =3.91, p=0.048*
20
T/T
n=34
16
C/T
n=146
12
C/C
n=129
8
2.4 104
2.6 104
2.8 104
3 104
3.2 104
Norepinephrine excretion, ng/gm
3.4 104
3.6 104
Online II
Human tyrosine hydroxylase (TH) promoter:
Graphical Observation of Linkage Disequilibrium (“GOLD”).
4 common SNPs (minor allele freq. 0.119-0.454) spanning 331 bp.
D’
D’
Promoter
position
5’ of ATG
Results by Lian Zhang in n=293 unrelated individuals of four different ancestries. 1-3-05.
Online IIIA
Human tyrosine hydroxylase (TH) promoter:
Graphical Observation of Linkage Disequilibrium (“GOLD”).
4 common SNPs (minor allele freq. 0.119-0.484) spanning 331 bp.
Promoter
position
5’ of ATG
D’
D’
D’
Results by Lian Zhang in n=189 unrelated individuals of European ancestry. 1-3-05.
Online IIIB
Human tyrosine hydroxylase (TH) promoter:
Graphical Observation of Linkage Disequilibrium (“GOLD”).
4 common SNPs (minor allele freq. 0.100-0.283) spanning 331 bp.
D’ D’
Promoter
position
5’ of ATG
Results by Lian Zhang in n=30 unrelated individuals of Asian ancestry. 1-3-05
Online IIIC
Human tyrosine hydroxylase (TH) promoter:
Graphical Observation of Linkage Disequilibrium (“GOLD”).
4 common SNPs (minor allele freq. 0.171-0.471) spanning 331 bp.
Promoter
position
5’ of ATG
D’
Results by Lian Zhang in n=35 unrelated individuals of Hispanic ancestry. 1-3-05.
Online IIID
Human tyrosine hydroxylase (TH) promoter:
Graphical Observation of Linkage Disequilibrium (“GOLD”).
4 common SNPs (minor allele freq. 0.090-0.410) spanning 331 bp.
D’
Promoter
position
5’ of ATG
Results by Lian Zhang in n=39 unrelated individuals of African ancestry. 1-3-05.
Online IIIE
Baroreceptor slope: Downward versus upward deflections.
Barorecepor slope, msec/mmHg
Downward deflections (logtda1)
A
4.00
Spearman correlation:
r =0.690, p<0.0001
3.00
A
A
A
2.00
AA
A
A
A
1.00
A
A
A
A A A
A
A
A
A
A
A
AA
A
A
A
A A
A
A
A
A A
A
A
A A A A
AA
AA A
A A
AA
A
A
A A A
A
AA A A
A
A A
A
A
A
AA
A A
A
AA A A
AA
A A
AA
A A
A
A
AA A AAA
AA A
A
A
A
AAA
A A AA A A
A
A
A A AA A
A A
A
A
A AA
A
AA A
A
A
A
A
A
A
A A A AA
A
A
A
AA
A
AA AA A
A
A
A
AA A
A A
A
A
A
A
A A
A
logtda1 = 0.70 + 0.63 * logtda2
R-Square = 0.50
A
0.00
A
1.00
2.00
3.00
4.00
Barorecepor slope, msec/mmHg
Upward deflections (lo gtda2)
Online IVA
TH pleiotropy.
Baroreceptor sensitivity as a determinant of basal norepinephrine release.
Stratification by TH promoter haplotype 2.
Tyrosine hydroxylase promoter haplotype 2: Pleiotropy.
Augmenting the coupling between the baroreflex
and catecholamine secretion in twins
2
4 10
Norepinephrine h =49.6+/-6.7%, p=0.0001*
Haplotype 2 on norepinephrine: p=0.0125*,
4.06% variation explained
4
2
Urinary norepinephrine, ng/gm
Baroreceptor downward slope h =43.0+/-7.3%, p<0.0001*
Haplotype 2 on baroreceptor upward slope: p=0.702
Pleiotropy: bivariate likelihood ratio test
2
χ =7.0, p=0.0082*
3.5 104
Haplotype 2
n=2 copies
(n=32 individuals)
3 104
Haplotype 2
n=1 copy
(n=164 individuals)
Haplotype 2
n=0 copies
(n=131 individuals)
2.5 104
2 104
5
10
15
20
Baroreceptor downward deflection slope, msec/mmHg
Downward deflections.
Online IVB