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Physiol. Genomics 19: 277-291, 2004. First published September 14, 2004; doi:10.1152/physiolgenomics.00151.2004
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Received 2 July 2004; accepted in final form 13 August 2004.
Physiological Genomics 19:277-291 (2004)
1094-8341/04 $5.00 © 2004 American Physiological Society

Functional allelic heterogeneity and pleiotropy of a repeat polymorphism in tyrosine hydroxylase: prediction of catecholamines and response to stress in twins

Lian Zhang 1, Fangwen Rao 1, Jennifer Wessel 2,4, Brian P. Kennedy 1, Brinda K. Rana 2,4, Laurent Taupenot 1, Elizabeth O. Lillie 1, Myles Cockburn 7, Nicholas J. Schork 2,4, Michael G. Ziegler 1 and Daniel T. O’Connor 1,3,4,5,6

1 Departments of Medicine, University of California at San Diego, San Diego, California
2 Psychiatry, University of California at San Diego, San Diego, California
3 Pharmacology, University of California at San Diego, San Diego, California
4 Polymorphism Research Laboratory, University of California at San Diego, San Diego, California
5 Center for Molecular Genetics, University of California at San Diego, San Diego, California
6 Veterans Affairs San Diego Healthcare System, San Diego, California
7 Department of Preventive Medicine, University of Southern California Keck School of Medicine, Los Angeles, California


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, has a common tetranucleotide repeat polymorphism, (TCAT)n. We asked whether variation at (TCAT)n may influence the autonomic nervous system and its response to environmental stress. To understand the role of heredity in such traits, we turned to a human twin study design. Both biochemical and physiological autonomic traits displayed substantial heritability (h2), up to h2 = 56.8 ± 7.5% (P < 0.0001) for norepinephrine secretion, and h2 = 61 ± 6% (P < 0.001) for heart rate. Common (TCAT)n alleles, particularly (TCAT)6 and (TCAT)10i, predicted such traits (including catecholamine secretion, as well as basal and poststress heart rate) in allele copy number dose-dependent fashion, although in directionally opposite ways, indicating functional allelic heterogeneity. (TCAT)n diploid genotypes (e.g., [TCAT]6/[TCAT]10i) predicted the same physiological traits but with increased explanatory power for trait variation (in contrast to allele copy number). Multivariate ANOVA documented genetic pleiotropy: joint effects of the (TCAT)10i allele on both biochemical (norepinephrine) and physiological (heart rate) traits. (TCAT)6 allele frequencies were lower in normotensive twins at genetic risk of hypertension, consistent with an effect to protect against later development of hypertension, and suggesting that the traits predicted by these variants in still-normotensive subjects are early, heritable, "intermediate phenotypes" in the pathogenetic scheme for later development of sustained hypertension. We conclude that common allelic variation within the tyrosine hydroxylase locus exerts a powerful, heritable effect on autonomic control of the circulation and that such variation may have implications in later development of cardiovascular disease traits such as hypertension.

microsatellite; cold pressor test; blood pressure; heart rate; hypertension


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
TYROSINE HYDROXYLASE CATALYZES the ring hydroxylation of L-Tyr to L-DOPA (dihydroxyphenylalanine), and is found in the cytosol of noradrenergic and dopaminergic neurons of the locus coeruleus, ventral tegmental area, and substantia nigra, as well as the adrenal medulla and sympathetic ganglia (39). The human tyrosine hydroxylase locus, on chromosome 11p15 (9), contains 13 exons/12 introns spanning ~8 kbp. Although a single-copy gene, tyrosine hydroxylase produces four different types of mRNA through alternative splicing of a single primary transcript (22), suggesting a novel means of regulating catecholamine levels.

This gene contains an informative simple sequence repeat (microsatellite) polymorphism consisting of the tetranucleotide repeat (TCAT)n, in 5–11 copies [(TCAT)5–11] within intron A (41). This microsatellite (sometimes abbreviated "HUMTH01") has been used to probe the role of tyrosine hydroxylase in psychiatric illnesses such as schizophrenia (31), and bipolar affective disease (32), yielding suggestive results (29, 31, 53). This microsatellite may function as a transcriptional enhancer, suggesting that the microsatellite may have a direct influence on gene expression (1, 30).

Essential hypertension is a multifaceted and progressive disorder spanning several decades of life. Differential (TCAT)n allele frequencies have been associated with hypertension (49) and blood pressure regulation (3, 24). The hemodynamic response to cold stress (the "cold pressor test") may be a predictor of development of later cardiovascular events, such as hypertension (25, 33, 46, 50, 54). Such responses, gauged even prior to the onset of disease, may be useful "intermediate phenotypes" in probing the genetic determination of disease traits such as hypertension (17, 37, 38).

Rapid release of catecholamines by the sympathetic nervous system is a primary neurohormonal mechanism mediating an organism’s stress response (6). Animal models exposed to acute and long-term cold stress have demonstrated increased gene expression of tyrosine hydroxylase (4, 42, 45, 51), the rate-limiting enzyme in catecholamine biosynthesis (18, 34); hence regulation of tyrosine hydroxylase is likely to be important for release of catecholamines in the human response to stress.

This study tested relationships among tyrosine hydroxylase (TCAT)n alleles, catecholamine secretion, and cardiovascular reactivity to stress. To probe the role of tyrosine hydroxylase polymorphism in stress-induced disease pathways, we turned to the classic human twin study design (5). Twins allow estimation of heritability (h2 = VG/VP), the contribution of additive genetic variance (VG) to phenotypic variance (VP) for any trait (15). Heritability gauges the tractability of any problem to genetic approaches, and twins then provide well-matched sibling pairs for further genetic association and linkage studies (5). In a large sample of twin pairs, we probed both biochemical and physiological autonomic phenotypes, including the hemodynamic responses to environmental (cold) stress (5, 38). We found substantial heritability of both biochemical (catecholamine secretion) and physiological (blood pressure and heart rate) autonomic traits. We then precisely determined the tyrosine hydroxylase (TCAT)n microsatellite diploid genotypes through bidirectional resequencing of intron A in each individual. Our results suggest that such autonomic traits are substantially heritable; that particular, high-frequency (TCAT)n alleles and diploid genotypes are functionally heterogeneous and predict such traits in allele copy number dose-dependent fashion; and that such alleles and genotypes act in pleiotropic fashion to jointly influence both biochemical and physiological responses.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Twin samples.
Twin recruitment proceeded by access to a population birth record-based twin registry (10), as well as by newspaper advertisement. We recruited n = 294 individuals from n = 148 twin pairs: n = 146 pairs plus 1 individual each from 2 additional twin pairs. There were n = 103 MZ pairs (M/M 21, F/F 82), and n = 45 DZ pairs (8 M/M, 30 F/F, and 7 M/F). Twin zygosity assignment was based on self-identification, with further confirmation by the presence or absence of heterozygosity at the (TCAT)n microsatellite (heterozygosity necessarily indicating dizygotic status; see below). The 294 subjects were all white (European ancestry); ethnicity was established by self-identification, as well as that for both parents and all 4 grandparents. Twin ages were 15 to 84 yr. Family histories for hypertension [in a first-degree relative before the age of 60 yr (37, 38)] were as follows: 64 pairs were positive (one or both parents); 69 pairs plus 1 singleton were negative; and 13 pairs plus 1 singleton were indeterminate/unknown. There were 264 individuals that were normotensive, and 30 were hypertensive (29 treated with antihypertensive medications; one untreated). None of the subjects had a history of renal failure. Definitions of subject characteristics are according to previous reports from our laboratory (38, 23). Subjects were volunteers from urban southern California (San Diego), and each subject gave informed, written consent; the protocol was approved by the Institutional Review Board. Descriptive statistics for these individuals are presented in Table 2.


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  		Table 2. Descriptive statistics for the twin study population

 
(TCAT)n polymorphism.
Genomic DNA from n = 294 phenotyped individuals was prepared from leukocytes in EDTA-anticoagulated blood, using PureGene extraction columns (Gentra Biosystems, Minneapolis, MN) as described (23). Public draft human (28) and mouse (55) "RefSeq" clone genome sequence was obtained from the UCSC Genome Bioinformatics web site (http://www.genome.ucsc.edu) and used as a scaffold for primer design and sequence alignment. The National Center for Biotechnology Information source clones for tyrosine hydroxylase base position numbers were NM_000360, NT_028310 or NT_009237 (chromosome 11p15), and NP_000351. PCR primers were designed by Primer3 or MacVector (Accelerys, San Diego, CA). For tyrosine hydroxylase intron A (TCAT)n resequencing, the sense oligonucleotide primer sequence was 5'-GGT GTT TGA GTC CCT GTT GG-3' and the antisense primer sequence was 5'-GTA CAC AGG GCT TCC GAG TG-3' (Integrated DNA Technologies, Coralville, IA). The amplicon size was 498 bp [for an amplicon surrounding (TCAT)10i]. Target sequences were amplified by PCR from 20 ng genomic DNA in a final volume of 25 µl, which also contained 0.5 U 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 model MJ PTC-225 thermal cycler, starting with 12 min of denaturation at 95°C, followed by 45 cycles of 95°C (denaturation) for 30 s, 61°C (annealing) for 1 min, 72°C (extension) for 1 min, and a final extension of 8 min at 72°C. A unique amplicon (single DNA band) was confirmed by ethidium bromide staining of the PCR product on agarose gels. PCR products were treated with exonuclease I and shrimp alkaline phosphatase to remove primers and dNTPs prior to cycle sequencing with BigDye terminators (Applied Biosystems). Sequence was determined, from each direction (both sense and antisense strands), on an ABI 3100 automated sequencer and analyzed using the Phred/Phrap/Consed suite of software to provide base quality scores (12, 13, 21, 57). Polymorphism and heterozygosity were detected using PolyPhred (35, 43) 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.

Single nucleotide polymorphism.
The Val81Met polymorphism in tyrosine hydroxylase (rs6356, A/G) was scored in a two-stage assay (7). In stage 1, PCR primers flanking the polymorphism were used to amplify the target region from 5 ng of genomic DNA. In stage 2, 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 vs. variant) was scored by MALDI-TOF mass spectrometry (low mass allele vs. high-mass allele). In n = 440 individuals ascertained from twin families, genotypic ratios were as follows: A/A, 72; A/G, 197; and G/G, 171. Allele frequencies were Met = A = 39% and Val = G = 61% (Hardy-Weinberg equilibrium {chi}2 = 1.41, P = 0.234).

Biochemical phenotyping: catecholamines.
Samples for measurement of plasma and urine catecholamines were quickly frozen at –70°C, prior to a sensitive radioenzymatic assay based on catechol-O-methylation (26). The assay uses a preconcentration step that increases sensitivity by ~10-fold over other catechol-O-methyltransferase-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 vivo.
Subjects were studied prospectively, before genotyping. Wild-type and variant subjects were studied during the same time interval. Blood pressure (in mmHg) and pulse interval (R-R interval or heart period, in ms/beat) were recorded continuously and noninvasively for 5 min in seated subjects with a radial artery applanation device and dedicated sensor hardware (Colin Pilot; Colin Instruments, San Antonio, TX) and software [ATLAS from WR Medical, Stillwater, MN; and Autonomic Nervous System/Tonometric Data Analysis (ANS/TDA) from Colin Instruments], calibrated every 5 min against ipsilateral brachial arterial pressure with a cuff sphygmomanometer. Heart rate was recorded continuously with thoracic EKG electrodes to the Colin Pilot. Vital signs were also recorded with the same devices during environmental stress, in the form of the cold pressor test (CPT; immersion of the left hand in ice water for 60 s after a preceding 10-min rest), as previously described (38, 40). We identified at least three beats with stable blood pressure and heart rate (each beat within ±10% of the mean value) just before and at the end of the cold stress.

Statistical analysis.
Descriptive statistics (means ± SE) were computed across all of the twins, using generalized estimating equations (GEE), in SAS (Statistical Analysis System, Cary, NC), to take into account intra-twin-pair correlations (11). Estimates of heritability (h2 = VG/VP, where VG is additive genetic variance and VP is total phenotypic variance) were obtained using the variance-component methodology implemented in the SOLAR ("sequential oligogenic linkage analysis routines") package (2), available at the SOLAR web site (http://www.sfbr.org/solar/). This method maximizes the likelihood assuming a multivariate normal distribution of phenotypes in twin pairs (monozygotic vs. dizygotic) with a mean dependent on a particular set of explanatory covariates. The null hypothesis (H0) of no heritability is tested by comparing the full model, which assumes genetic variation (VG), and a reduced model, which assumes no genetic variation, using a likelihood ratio test. All heritability estimates were adjusted for age and sex, because of the effects of these covariates on several traits (Table 2). SOLAR was also used to evaluate whether allelic variation at the locus contributed to a significant fraction of the trait heritability (i.e., locus-specific VG), by comparing models including or excluding the genotype as a covariate. Each study subject was categorized according to carrier status for particular (TCAT)n alleles (2, 1, or 0 copies of the allele). To maximize statistical power, only the two most common (TCAT)n alleles, (TCAT)6 and (TCAT)10i, or their diploid genotypic combinations, were so analyzed. During allele, haplotype, and haplotype pair (diplotype) associations, multivariable analyses were carried out to control for confounding by age and sex (as covariates), because of their effects on several of the biochemical and physiological phenotypes (Table 2).

Multivariate ANOVA (MANOVA) tested for joint effects of one independent variable (genotype) on two dependent variables (e.g., biochemical and physiological). For MANOVA, both individuals of each DZ pair, but only one individual from each MZ pair, were evaluated. Data were stored in Microsoft Access, and analyses were conducted in SPSS (Statistical Package for the Social Sciences, Chicago, IL), SAS, or SOLAR.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Tyrosine hydroxylase (TCAT)n genomics.
We resequenced the (TCAT)n microsatellite and adjacent regions in the amplified tyrosine hydroxylase intron A from n = 294 of the phenotyped individuals. We identified a total of 6 different alleles, generated by 6–10 repetitions of the core tetranucleotide motif TCAT: (TCAT)6, (TCAT)7, (TCAT)8, (TCAT)9, and 10-repeat alleles exhibiting two sequence variants, the perfect repeat (TCAT)10 and a (TCAT)4CAT(TCAT)5 imperfect repeat, also known as (TCAT)10i. Figure 1 shows actual sequence profiles from several specific common diploid combinations: (TCAT)6 homozygotes, (TCAT)6/(TCAT)10i heterozygotes, and (TCAT)10i homozygotes. Sequencing from the opposite (antisense) direction confirmed each genotype.



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  		Fig. 1. Determination of (TCAT)n genotype by resequencing a 475-bp amplicon in tyrosine hydroxylase intron A from human genomic DNA. The ABI 3100 sequence trace data display window from PolyPhred is shown, along with the direction of sense-strand sequencing (5' -> 3'). Results are shown for a (TCAT)6/(TCAT)6 homozygote, a (TCAT)10i/(TCAT)10i heterozygote, and a (TCAT)6/(TCAT)10i homozygote. (TCAT)10i is the (TCAT)4CAT(TCAT)5 imperfect repeat. Sequencing from the opposite (antisense) direction confirmed each genotype.

 
We resequenced the amplified (TCAT)n region from nine chimpanzees to determine the likely ancestral allele(s) at the polymorphic site; seven chimpanzees were (TCAT)6/(TCAT)6 homozygotes, while two were (TCAT)6/(TCAT)7 heterozygotes. One gorilla was a (TCAT)4/(TCAT)6 heterozygote, while one orangutan was a (TCAT)4/(TCAT)4 homozygote. Since the chimpanzee is the extant non-human primate with the closest evolutionary ties to humans [divergent lineages ~4–6 million years ago (14)], it is conceivable that the ancestral alleles in humans are (TCAT)6 or (TCAT)7, with the additional human alleles [5–10 (TCAT)n repeats] occurring later within the human lineage, although this conclusion is tentative.

Human (TCAT)n allele frequencies are presented in Table 1A, while diploid genotype frequencies are shown in Table 1B. The allele frequencies are similar to those previously reported in the literature (41), and the diploid genotype frequencies did not differ significantly from expectation under Hardy-Weinberg equilibrium ({chi}2 = 15.9, df = 18, P = 0.5974).


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  		Table 1. Tyrosine hydroxylase (TCAT)n genomics: Allele frequencies and diploid genotypes

 
Autonomic phenotypes: descriptive statistics and effects of age and sex.
Table 2 describes the subject population (n = 294 individuals). Females (n = 231) had somewhat higher poststress heart rate (P = 0.0291) and heart rate change (P = 0.0293) values than males (n = 63), as well as lower basal plasma epinephrine values (P = 0.0092), consistent with previous reports (19).

Older subjects (age ≥40 yr) had higher basal systolic blood pressure (SBP) (P < 0.0001) and diastolic BP (DBP) (P < 0.0001), as well as poststress SBP (P < 0.0001) and DBP (P = 0.0001) values, than younger subjects. Urinary norepinephrine excretion was increased (P < 0.0001) in older subjects.

Phenotype distributions and interindividual correlations: autonomic biochemistry and physiology.
Figure 2 shows frequency histograms of basal catecholamine secretion, as well as cold stress-induced changes in SBP, DBP, and heart rate in the twin group. The distributions seemed to be unimodal in each case, often with skewing toward higher values. Traits with such distributions are likely to be tractable to parametric statistical analyses (e.g., ANOVA, regression), such as those undertaken here.



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  		Fig. 2. Frequency distributions of biochemical and physiological autonomic phenotypes in the twin population. Also shown are normal curves fitted to the data. The number of histogram intervals (bins) is 14 (the closest integral number to the square root of 192). For these histograms, we plotted both members of each DZ pair but only one member of each MZ pair. A: norepinephrine excretion (uNorepinephrine) in ng/g. B: epinephrine excretion (uEpinephrine) in ng/g. C: plasma norepinephrine (pNorepinephrine) in pg/ml. D: plasma epinephrine (pEpinephrine) in pg/ml. E–G: blood pressure and heart rate change ({Delta}) in response to cold stress. E: change in systolic blood pressure ({Delta}SBP, mmHg). F: change in diastolic BP ({Delta}DBP, mmHg). G: change in heart rate ({Delta}HR, beats/min).

 
Table 3 presents interindividual correlations between variables: correlations above and to the right of the diagonal are parametric (Pearson), while those below and to the left of the diagonal are nonparametric (Spearman). In general, similar correlations were obtained with both methods. Because of the effects of sex and age on several of the biochemical and physiological traits (Tables 2 and 3), further inferential statistics (SOLAR and GEEs) were performed on age- and sex-adjusted data.


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  		Table 3. Correlations among demographic, physiological, and biochemical variables in the twins

 
Figure 3 illustrates two significant correlations between biochemical and physiological traits in this population, with mechanistic implications for autonomic control of blood pressure. Figure 3A shows the positive effect of renal norepinephrine excretion on resting SBP (Pearson r = 0.48, r2 = 0.23, n = 111, P < 10–7), while Fig. 3B illustrates the positive relationship between renal norepinephrine excretion and cold stress-induced increment in SBP (r = 0.30, r2 = 0.09, n = 112, P = 0.001).



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  		Fig. 3. Trait correlations: biochemical and physiological. Prediction of resting systolic blood pressure (SBP; A) and cold stress-induced change in SBP (B) by renal norepinephrine excretion (ng norepinephrine/g creatinine) in twins. For these regressions, we plotted only one member of each pair. A: positive effect of renal norepinephrine excretion on resting SBP. B: positive relationship between renal norepinephrine excretion and cold stress-induced increment in SBP.

 
Autonomic phenotypes: heritability (h2).
Both plasma catecholamine concentrations and renal/urinary catecholamine excretions were significantly heritable (Table 4), with the most prominent values for plasma norepinephrine (h2 = 56.8 ± 7.5%, P < 0.0001) and urinary epinephrine excretion (h2 = 47.1 ± 8.8%, P < 0.0001).


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  		Table 4. Heritability (h2 = VG/VP) of autonomic function in twins: biochemical and physiological traits

 
Basal blood pressure and heart rate displayed significant heritability, with heart rate substantially more heritable (at h2 = 61 ± 6%, P < 0.0001) than either SBP (h2 = 26 ± 8%, P = 0.0016) or DBP (h2 = 18 ± 9%, P = 0.0359). Stress-induced changes in vital signs were also heritable, whether expressed as maximal values, absolute changes (maximal minus basal), or percent changes. The h2 values for stress-induced changes were noticeably greater than the corresponding basal values: for poststress DBP, h2 = 37 ± 8 (vs. 18 ± 9% for basal DBP); and for change in DBP ({Delta}DBP), h2 = 32 ± 8%.

Observed values for h2 of traditionally highly heritable reference traits (weight at h2 = 87 ± 2%, P < 0.0001; height at h2 = 93 ± 1%, P < 0.0001) in our sample are consistent with previous observations (15), confirming the reliability of this method for estimating heritability in our twin sample.

Association of (TCAT)n alleles with autonomic biochemistry and physiology.
Table 5 illustrates the effect of particular (TCAT)n alleles on autonomic traits. As an initial approach, and to maximize statistical power, only the most common alleles (Table 1) are shown: (TCAT)6 or (TCAT)10i. We used the variance components method in SOLAR to test whether allelic variation at (TCAT)n contributed significantly to trait heritability (h2 = VA/VP). To evaluate the effect of particular alleles, trait means (by GEEs) were grouped by the number of copies of the particular allele (0, 1, or 2 copies). The h2 method documented significant effects of (TCAT)n alleles on basal blood pressure and heart rate, poststress heart rate, and catecholamine secretion.


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  		Table 5. Effects of common (TCAT)n alleles on biochemical and physiological traits in the twins

 
(TCAT)6 copy number affected basal pulse interval (P = 0.007) and heart rate (P = 0.0003) and poststress heart rate (P = 0.0001). (TCAT)10i copy number affected basal pulse interval (P = 0.0349), plasma epinephrine (P = 0.0133), and renal norepinephrine excretion (P = 0.0309).

Figure 4 illustrates these (TCAT)n allelic effects. Increasing (TCAT)6 copy number increased pulse interval (Fig. 4A), as well as both basal and poststress heart rate (Fig. 4B). By contrast, increasing (TCAT)10i copy number decreased pulse interval (Fig. 4C) while increasing renal norepinephrine excretion (Fig. 4D). Of note, (TCAT)6 and (TCAT)10i copy numbers exerted directionally opposite effects on pulse interval (Fig. 4E).



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  		Fig. 4. (TCAT)n alleles: effects on biochemical and physiological phenotypes in twins. Results are shown as the effect of the number of copies of that allele per diploid genome (0, 1, or 2 copies). Mean values ± 1 SE are shown, calculated from all individuals (both members of each twin pair, MZ and DZ) in that group, by generalized estimating equations (GEE). A: effect of (TCAT)6 on pulse interval (cardiac R-R interval). SOLAR P = 0.007, explaining 3.6% of trait variation. B: effect of (TCAT)6 on heart rate (HR) pre- and post-cold stress. For prestress, SOLAR P = 0.0003, explaining 5.7% of trait variation. For poststress, SOLAR P = 0.0001, explaining 5.4% of trait variation. C: effect of (TCAT)10i on pulse interval (cardiac R-R interval). SOLAR P = 0.0349, explaining 2.1% of trait variation. D: effect of (TCAT)10i on renal norepinephrine excretion. SOLAR P = 0.0309, explaining 1.4% of trait variation. E: functional allelic heterogeneity: directionally opposite effects of (TCAT)6 and (TCAT)10i alleles on pulse interval (cardiac R-R interval). For (TCAT)6, SOLAR P = 0.007, explaining 3.6% of trait variation. For (TCAT)10i, SOLAR P = 0.0349, explaining 2.1% of trait variation.

 
The less common (TCAT)n alleles [Table 1, 0.5–17.4%: (TCAT)7, (TCAT)8, (TCAT)9, and (TCAT)10] did not significantly affect any or the traits in these analyses (all P > 0.1); however, we cannot exclude the possibility that a larger sample size n might have been required to discern significant effects of these relatively unusual alleles. Nonetheless, inability to discern any trait effects during allelic associations in n = 294 individuals (n = 588 chromosomes) speaks against a frequent effect for any such allele in the general population (44, 48).

(TCAT)n diploid genotypes: effects on autonomic phenotypes.
Diploid genotypes composed of (TCAT)6 and (TCAT)10i alleles were tested for their effects on the phenotypes (Table 6): (TCAT)6/(TCAT)6 homozygotes, (TCAT)6/(TCAT)10i heterozygotes, and (TCAT)10i/(TCAT)10i homozygotes. These diploid combinations were chosen because they were common enough to enable adequate statistical power in this sample of n = 294 genotyped individuals (Table 1). Once again we tested a contribution of allelic variation at the locus to trait heritability (h2 = VA/VP) by SOLAR and GEEs (Table 6), documenting effects of (TCAT)n diploid genotype on basal and poststress heart rate and catecholamine secretion.


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  		Table 6. Effect of (TCAT)n diploid genotype ("diplotype") on autonomic phenotypes

 
(TCAT)6/(TCAT)10i genotypes influenced basal pulse interval (P = 0.0472), as well as basal (P = 0.0305) and poststress (P = 0.0011) heart rate (Table 6).

Figure 5 illustrates the effects of these diploid genotypes on autonomic traits: genotypic influences upon pulse interval (Fig. 5A) and heart rate pre- and poststress (Fig. 5B). Increasing copy number of (TCAT)10i [or decreasing number of (TCAT)6] alleles resulted in a fall in basal pulse interval (i.e., a rise in heart rate; Fig. 5, A and B), as well as a rise in poststress heart rate (Fig. 5B).



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  		Fig. 5. (TCAT)6/(TCAT)10i diploid genotypes ("diplotypes"): effects on physiological phenotypes in twins. Mean values ± 1 SE are shown, calculated from all individuals (both members of each twin pair, MZ and DZ) in that group, by GEE. A: effects of genotype on basal pulse interval (cardiac R-R interval). SOLAR P = 0.0472, explaining 13.0% of trait variation. B: effects of genotype on pre- and poststress heart rate. For prestress, SOLAR P = 0.0305, 13.2%. For poststress, SOLAR P = 0.0011, explaining 15.3% of trait variation.

 
Pleiotropic phenotypic clusters: joint predictions of autonomic biochemistry and physiology by particular (TCAT)n alleles or diploid genotypes.
We used MANOVA to test whether (TCAT)n variants jointly influenced both biochemical and physiological traits within the autonomic system, suggesting pleiotropy. In this scheme, a single independent variable (genotype) is simultaneously tested for effects on two dependent variables: biochemical (e.g., norepinephrine secretion) and physiological (e.g., pulse interval).

Figure 6 illustrates the pleiotropic effects of the (TCAT)10i polymorphism. Increasing numbers of (TCAT)10i alleles (0, 1, or 2 copies) were associated with a progressive and parallel increase in renal norepinephrine excretion, coupled with a decrease in pulse interval (MANOVA: Pillai F = 3.30, P = 0.012).



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  		Fig. 6. Pleiotropy and phenotypic clusters. (TCAT)10i joint predictions of biochemical (renal norepinephrine excretion, NE) and physiological (pulse interval, PI) phenotypes in twins. Clusters are displayed as mean values ± 1 SE for each trait, by GEE. Joint prediction of two traits by one genotype was tested by multivariate ANOVA (MANOVA), wherein the model contains one independent variable (genotype) and two dependent variables (biochemical and physiological). MANOVA: Pillai trace F = 3.30, P = 0.012; Wilks lambda F = 3.38, P = 0.011; Hotelling trace F = 3.46, P = 0.009; Roy largest root F = 7.054, P = 0.001.

 
The (TCAT)n polymorphism and genetic risk of hypertension.
Our sample had too few patients with hypertension for meaningful genotype associations. However, among the twins, there were substantial numbers at genetic risk of hypertension, by virtue of positive family history in a first-degree relative (Table 7) (36, 38). Among all n = 294 genotyped individuals, n = 128 were family history positive, n = 139 were negative, and n = 27 were indeterminate. We tested whether common (TCAT)n alleles (or genotypes) differed in these genetic risk groups. When the family history groups were stratified by presence or absence of the (TCAT)6 allele, the frequencies differed significantly ({chi}2 = 4.29, P = 0.0384). (TCAT)10i stratification (P = 0.3535) or (TCAT)6/(TCAT)10i genotype (P = 0.2877) groups did not differ by family history.


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  		Table 7. (TCAT)n common allele and diploid genotype frequencies in individuals stratified by genetic risk (family history) of hypertension

 
Tyrosine hydroxylase single nucleotide polymorphism.
The biallelic Val81Met polymorphism was also scored in these individuals, and the genotypes were in Hardy-Weinberg equilibrium ({chi}2 = 1.41, P = 0.234). However, allelic variation at this single nucleotide polymorphism locus (in our subjects: Val = 61%, Met = 39%) did not associate with any of the biochemical or physiological phenotypes (all P > 0.1).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Tyrosine hydroxylase polymorphisms.
Tyrosine hydroxylase is the rate-limiting enzyme in catecholamine biosynthesis (17, 18). Profound tyrosine hydroxylase deficiency in humans, as occurs after unusual inactivating mutations (Leu205Pro or Gln381Lys) in both alleles at the locus, results in widespread disturbance of neuropsychiatric function, such as autosomal recessive, L-DOPA-responsive dystonia (17, 18). Complete homozygous ablation of the tyrosine hydroxylase locus, by homologous recombination-directed gene targeting in transgenic mice, is lethal by the early postnatal period (8). A common biallelic variant of tyrosine hydroxylase in coding region exon 2 (Val81Met, allelic ratios in our subjects: Val = 61%, Met = 39%) is of uncertain functional significance, since amino acid residue 81 lies far from the catalytic domain of the enzyme (amino acid residues 187 through 455), as established by X-ray crystallography (18, 20).

In humans, more subtle, perhaps regulatory genetic alterations in tyrosine hydroxylase may have functional consequences. The (TCAT)n tetranucleotide repeat (sometimes abbreviated "HUMTH01") in tyrosine hydroxylase intron A has been the subject of a small number of studies, both clinical disease associations (3, 24, 27, 31, 32, 49, 53) and in vitro functional tests of transcriptional regulation (1, 30).

Differential (TCAT)n allele frequencies have been associated with hypertension(49), blood pressure regulation (3, 24), schizophrenia (27, 31), and bipolar affective disease (32). Barbeau et al. (3) found that (TCAT)6 and (TCAT)10i alleles seemed to be cardioprotective by association with attenuation of the hemodynamic response to stress with increasing age, whereas (TCAT)7 seemed to be deleterious by association with higher resting SBP and greater hemodynamic response to stress with increasing body mass index. Sharma et al. (49) found a lower frequency of the (TCAT)9 allele in normotensive subjects, accompanied by lower plasma norepinephrine, whereas Wei et al. (56) found (TCAT)9 to be associated with significantly higher norepinephrine levels. The (TCAT)n motif may bind such transcription factors as Fos/Jun (30) or ZNF191 (1) and may function as a transcriptional enhancer when tested in transfected/expressed promoter/reporter plasmids (1, 30). However, the effects of this putative enhancer are not well understood: when fused to a heterologous (thymidine kinase) promoter, the (TCAT)n motif seems to activate transcription (30), whereas fusion to the homologous (tyrosine hydroxylase) promoter seems to repress transcription (1). In the homologous system, all (TCAT)n length variants tested repress transcription, in copy number-dependent fashion (1).

Our studies of the (TCAT)n polymorphism offered two particular advantages. The first is precision of genotyping. By carefully resequencing the (TCAT)n region, we were able to assign precise allele scores in each individual (Fig. 1). Absent sequencing, the distinction between (TCAT)10 and (TCAT)10i is difficult, and these two alleles have not been well discriminated in some studies (49). The second advantage is power. We established (TCAT)n alleles and genotypes in n = 294 individuals (with n = 588 chromosomes) who had undergone careful biochemical and physiological phenotyping. This relatively large pool of alleles and genotypes (Table 1) allowed us to accrue enough allele- and genotype-stratified individuals to begin to discern effects of the most common alleles [(TCAT)6 and (TCAT)10i] on the traits under study.

Twin phenotype interindividual correlations: biochemistry vs. physiology.
Several of the biochemical and physiological phenotypes were significantly correlated between/among individuals (Table 3). Particularly striking (Fig. 3) was the prediction by renal norepinephrine excretion of basal SBP (Pearson r = 0.48, r2 = 0.23, n = 111, P < 10–7), as well as stress-induced SBP increment (r = 0.30, r2 = 0.09, n = 112, P = 0.001). Indeed, for basal SBP, r2 = 0.23 indicates that ~23% of the interindividual variance in SBP might be accounted for by corresponding variability in sympathetic tone. These results suggest that sympathetic tone is a strong determinant of interindividual differences in basal and stress blood pressure and raise the possibility that functional polymorphism at tyrosine hydroxylase might influence not only sympathetic tone, but also basal and stress-augmented blood pressures.

Twin phenotypes and heritability (h2): autonomic biochemistry and physiology.
Twin studies allow the estimation of heritability (h2 = VG/VP) of any trait, where h2 is the fraction of trait variance (VP) accounted for by additive genetic variance (VG) (15); substantial heritability is a prerequisite for the ultimate tractability of any trait to further genetic analysis (15). Indeed, both the biochemical and physiological traits studied here demonstrated significant heritability (Table 4). Particularly heritable in our twin sample were the biochemical traits of plasma norepinephrine concentration (h2 = 56.8 ± 7.5%, P < 0.0001) and renal epinephrine excretion (h2 = 47.1 ± 8.8%, P < 0.0001). These results demonstrate somewhat greater heritability for catecholamine secretion than previous reports (16), perhaps because the O-methylation-based radioenzymatic catecholamine assays we employ are substantially more sensitive than older assays, enabling more precise quantification of catecholamines (especially epinephrine) at their very low (~fM) circulating concentrations (26).

Among physiological traits, pulse interval (reciprocal of heart rate) was strikingly heritable after prolonged (5 min) monitoring (h2 = 61 ± 6%, P < 0.0001); heart rate just before (h2 = 54 ± 7%, P < 0.0001) and at the conclusion (h2 = 52 ± 6%, P < 0.0001) of cold stress also displayed relatively high heritability. Within blood pressure traits, baseline SBP (h2 = 26 ± 8%, P = 0.0016) and DBP (h2 = 18 ± 9%, P = 0.0359) were heritable, while poststress DBP (h2 = 37 ± 8%, P < 0.0001) was substantially more heritable than the baseline value.

"Intermediate phenotypes."
Autonomic traits of relatively high heritability (Table 4; such as catecholamine secretion, baseline pulse interval, or poststress DBP) may be of particular value in investigation of the genetic underpinnings of hypertension, a complex trait with both genetic and environmental determinants, and likely even multiple genetic (or "polygenic") determinants (17, 36, 38, 46, 52). Such traits may be valuable "intermediate phenotypes" (17, 36, 38, 46, 52) for hypertension, especially if they display greater heritability then the ultimate disease trait (blood pressure), can be elicited in still-normotensive individuals, and suggest testable candidate genetic loci (such as tyrosine hydroxylase). Several of the phenotypes studied here (Table 4) meet each of these criteria: catecholamine secretion (up to h2 = 56.8 ± 7.5%, P < 0.0001), pulse interval (h2 = 61 ± 6%, P < 0.0001) and heart rate, as well as poststress DBP (h2 = 37 ± 8%, P < 0.0001). The hemodynamic response to cold stress (the "cold pressor test") may be a predictor of development of later cardiovascular events, such as hypertension (47, 50, 54). We thus went on to explore genetic influences on such autonomic traits (biochemical and physiological), beginning with common polymorphisms at the tyrosine hydroxylase locus.

(TCAT)n allelic associations: Pleiotropy and "intermediate phenotypes."
To explore the effects of particular (TCAT)n alleles on these traits, we stratified each individual on the basis of number of copies per genome (0, 1, or 2) of the allele in question (Table 5, Fig. 4). To maximize statistical power, we focused our efforts on the most common alleles (Table 1): (TCAT)6 and (TCAT)10i. The results seemed to support a conclusion of pleiotropic effects of tyrosine hydroxylase (TCAT)n polymorphisms (i.e., one gene -> many phenotypes).

(TCAT)10i copy number influenced a number of autonomic traits, both biochemical and physiological (Table 5, Fig. 4): plasma epinephrine concentration, renal norepinephrine excretion, and basal pulse interval. What underlies such pleiotropic effects? A potential mechanistic clue comes from the trait correlations (Table 3, Fig. 3): renal norepinephrine excretion influenced both basal and stress-augmented blood pressure. Thus the pleiotropic effects of (TCAT)10i likely represent a mechanistic chain of events (or "intermediate phenotypes") (Fig. 7): gene [(TCAT)10i] -> biochemical trait (norepinephrine production) -> physiological traits. This cascade is in accordance with a chain of "intermediate phenotypes" of graded proximity to gene action (Fig. 7) (17, 36).



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  		Fig. 7. Tyrosine hydroxylase (TCAT)n polymorphism and "intermediate phenotypes" in the autonomic system. In the "intermediate phenotype" schema (36), biochemical traits are postulated to be determined earlier and more proximately by genotype than are physiological traits. TH, tyrosine hydroxylase; BP, blood pressure; HR, heart rate; {Delta}, change.

 
The notion of pleiotropy is reinforced by biochemical and physiological trait clustering when stratified by genotype (Fig. 6). We formally tested the joint effects of particular genotypes on combinations of two phenotypes (Fig. 6) by MANOVA. Significant effects of (TCAT)10i copy number were noted for the combination of renal norepinephrine excretion with pulse interval (Fig. 6).

Functional allelic heterogeneity at (TCAT)n.
(TCAT)6 and (TCAT)10i seemed to influence both biochemical and physiological autonomic traits (Table 5, Fig. 4). Yet some traits (such as pulse interval) were influenced in directionally opposite effects by (TCAT)6 vs. (TCAT)10i (Fig. 4E). This seems to represent qualitatively different effects of these two alleles, rather than simple unidirectional quantitative trait variation. Is this compatible with the in vitro effects of the (TCAT)n polymorphism on transcription? When fused to the homologous (tyrosine hydroxylase) promoter and transfected into chromaffin cells, (TCAT)n monotonically decreases promoter activity, in copy number-dependent fashion [from (TCAT)5 to (TCAT)10]. Thus the previously described in vitro effects of the (TCAT)n motif on transcription cannot explain the directional in vivo phenotypic associations of (TCAT)10i or (TCAT)6 (e.g., Fig. 4E).

Strength of effect of (TCAT)n on autonomic phenotypes: percent trait determination.
The strength of effect of polymorphisms on traits was be assessed by SOLAR, indicating the fraction of trait variation explained by allelic variation at the locus (Figs. 4 and 5). Trait variance determination ranged up to 15.3%, for (TCAT)n diploid genotypes on poststress heart rate (Fig. 5B). Thus (TCAT)n variation seems to exert a substantial effect on human autonomic function.

Inspection of the percent trait determination values revealed evidence of more prominent effects for particular combinations of genotypes and phenotypes: values were generally greater for diploid genotype associations (Table 6, Fig. 5) than for allele copy number associations (Table 5, Fig. 4). For example, for pulse interval or heart rate, diploid genotype determinations of trait ranged from 13.0–15.3%, while allele determinations were more modest, at 2.1–5.7%. Perhaps this is not surprising, in that stratification by diploid genotype provides more precise and comprehensive (indeed, complete) information about each individual’s (TCAT)n locus; by contrast, allele copy number (0, 1, or 2 copies) provides complete information only when copy number n = 2.

Implications for human biology and disease.
We have presented evidence that allelic variation at the tyrosine hydroxylase locus exerts widespread determination of phenotypic variation in the autonomic nervous system. The phenotypic pleiotropy (Fig. 6) documented for the effects of (TCAT)n alleles to jointly predict both biochemical and physiological traits is consistent with the "intermediate phenotype" hypothesis (Fig. 7) (36), wherein both biochemical traits (such as norepinephrine secretion) and more distal physiological traits (such as heart rate or blood pressure) flow from the action of the same disease predisposition alleles. Such "intermediate phenotypes" may be advantageous in the study of complex traits such as hypertension, because they can be scored even in the still-normotensive offspring of hypertensive probands, and may suggest not only logical candidate genetic loci (such as tyrosine hydroxylase), but also early pathophysiological mechanisms (such as catecholamine secretion) whereby risk alleles act to influence the ultimate disease trait (36).

Do our results in generally healthy twins have implications for the later development of disease states such as hypertension? We documented the heritability of blood pressure (Table 4) and noted that (TCAT)6 allele frequencies differed between twins stratified by genetic risk (family history) of hypertension: in particular, family history-positive individuals were less likely to bear the (TCAT)6 allele ({chi}2 = 4.29, P = 0.0384; Table 7). Since increasing (TCAT)6 allele copy number is associated with lower basal and stress-induced heart rates (Table 5, Fig. 4), our results suggest a mechanism whereby (TCAT)6 alleles may be protective against the future development of hypertension.

A previous study of the (TCAT)n repeat by Sharma et al. (49) found allele and genotype frequency differences in hypertension, wherein the frequency of the longest alleles [their allele "E", corresponding to a group including our (TCAT)10 plus (TCAT)10i] was increased in patients with hypertension; however, the level of resolution of genotyping in that study (49) did not discriminate between the (TCAT)10 and (TCAT)10i alleles. Nonetheless, it is noteworthy that we found positive effects of increasing copy number of (TCAT)10i on basal and stress-induced heart rate (Table 5, Fig. 4) as well as plasma epinephrine and renal norepinephrine excretion (Table 5, Fig. 4). Thus our results complement those of Sharma et al. (49) and provide a mechanistic basis for such observations in hypertension.

Is the (TCAT)n polymorphism itself responsible for the phenotypic associations we observed in vivo? In vitro, the (TCAT)n motif may bind trans-acting factors and function as a transcriptional enhancer, when ligated to heterologous or homologous promoter regions in transfected promoter/reporter plasmids (1, 30). However, these effects are not completely understood, may be directionally opposite in different cell types and with different core promoters, and seem to be unidirectional and dose dependent for increasing (TCAT)n copy number (1, 30). By contrast, we observed directionally opposite effects for the (TCAT)6 and (TCAT)10i alleles on adrenergic phenotypes in vivo (Fig. 4B). Thus we suspect that the emerging biology being described for the (TCAT)n motif in vitro (1, 30) cannot explain the (TCAT)n associations we have observed in vivo.

If the (TCAT)n motif itself is not causative in the observed trait associations, perhaps the (TCAT)n region is in linkage disequilibrium with another, nearby, causative variant. The inactivating Mendelian variants Leu205Pro and Gln381Lys are far too rare to account for the common associations found here (17). The Val81Met variant studied here was quite common (minor allele frequency 39%) but is of uncertain function and was not associated with variation in the autonomic traits in these subjects. The tyrosine hydroxylase locus has not yet be the target of systematic variant discovery in healthy individuals; thus additional common variants (either coding or regulatory) may remain to be discovered.

Are there genetic loci other than TH itself that might influence tyrosine hydroxylase activity in vivo? The enzyme GTP cyclohydrolase 1 (GCH1) is rate-limiting in provision of the pterin cofactor to tyrosine hydroxylase (17); indeed, autosomal recessive deficiency of GCH1 may produce a clinical syndrome of L-DOPA-responsive dystonia similar to that seen in autosomal recessive tyrosine hydroxylase deficiency. Alternatively, variation in the tyrosine hydroxylase transactivator polypeptide YWHAQ might change enzymatic activity posttranslationally.


   GRANTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by the Department of Veterans Affairs and the National Institutes of Health.


   ACKNOWLEDGMENTS
 
We appreciate the assistance of the General Clinical Research Center and its core laboratory. The California Twin Program assisted us in ascertaining twins from its population-based registry. Several individuals assisted us in twin phenotyping: Peter Cadman, Rubin Chandran, Danuta King, and Shagun Chopra.


   FOOTNOTES
 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: D. T. O’Connor, M.D., Dept. of Medicine (9111H), UCSD School of Medicine, 3350 La Jolla Village Drive, San Diego, CA 92161 (E-mail: doconnor{at}ucsd.edu; web site: http://medicine.ucsd.edu/hypertension).


   REFERENCES
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
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