Molecular Psychiatry (1999) 4, 360–368
 1999 Stockton Press All rights reserved 1359–4184/99 $15.00
ORIGINAL RESEARCH ARTICLE
Identification of four variants in the tryptophan
hydroxylase promoter and association to behavior
A Rotondo1,3, KE Schuebel4, AW Bergen3,5, R Aragon3, M Virkkunen6, M Linnoila3,✠, D Goldman3
and DA Nielsen2
1
Department of Psychiatry, Neurobiology, Pharmacology, and Biotechnologies, University of Pisa, 56100 Pisa, Italy;
Section of Molecular Genetics, 3Laboratory of Neurogenetics, National Institute on Alcoholism and Alcohol Abuse,
National Institutes of Health, Rockville, MD 20852; 4Johns Hopkins Oncology Center, Baltimore, MD 21231; 5Genetic
Epidemiology Branch, National Cancer Institute, Rockville, MD 20852, USA; 6Department of Psychiatry, University of
Helsinki, Helsinki, Finland
2
One of the most replicated findings in biological psychiatry is the observation of lower 5hydroxyindoleacetic acid concentrations, the major metabolite of serotonin, in the brain and
cerebrospinal fluid of subjects with impulsive aggression. Tryptophan hydroxylase (TPH) is
the rate-limiting enzyme in the synthesis of serotonin, however functional variants have not
been reported from the coding sequence of this gene. Therefore, we screened the human TPH
promoter (TPH-P) for genetic variants which could modulate TPH gene transcription. The TPHP (2093 nucleotides) was screened for sequence variation by SSCP analysis of 260 individuals
from Finnish, Italian, American Caucasian, and American Indian populations. Four common
polymorphisms were identified: −7180T⬎G, −7065C⬎T, −6526A⬎G, and −5806G⬎T
(designated as nucleotides upstream of the translation start site). In the Finns, the four polymorphisms had a minor allele frequency of 0.40 and in this population linkage disequilibrium
between the four loci was complete. In the other populations the minor allele frequencies
ranged from 0.40 to 0.45. TPH −6526A⬎G genotype was determined in 167 unrelated Finnish
offenders and 153 controls previously studied for the TPH IVS7+779C⬎A polymorphism. A
significant association was observed between −6526A⬎G and suicidality in the offenders. TPH
−6526A⬎G and the previously reported intron seven polymorphism, TPH IVS7+779C⬎A, exhibited a normalised linkage disequilibrium of 0.89 in Finns. Normalized linkage disequilibrium
was reduced in other populations, being 0.49 and 0.21 in Italians and American Indians,
respectively. In conclusion, four TPH-P variants were identified which can be used for haplotype-based analysis to localize functional TPH alleles influencing behavior.
Keywords: tryptophan hydroxylase; serotonin; promoter; genetic polymorphisms; suicidality;
aggression; variant
Introduction
The neurotransmitter serotonin is involved in a variety
of brain functions, including mood, arousal, sleep, food
intake, pain perception, temperature regulation,
aggression, sexual behavior, neuroendocrine functions,
and cognitive processes. Because of the multiple roles
of serotonin, it is not surprising that alterations in serotonin biosynthetic pathways have been postulated in
the pathogenesis of several mental disorders, for
example mood disorders, anxiety disorders, eating disorders, migraine, and disorders of impulse control,
such as suicidality.1
One of the most replicated findings in biological psychiatry has been the reduced level of cerebral fluid
Correspondence: A Rotondo, MD, Dept of Psychiatry, Neurobiology, Pharmacology and Biotechnologies, University of Pisa, via
Roma 67, 56100 Pisa, Italy. E-mail: a.rotondo얀psico.med.unipi.it
✠Deceased
Received 26 February 1999; revised and accepted 18 May 1999
(CSF) 5-hydroxyindoleacetic acid (5-HIAA) in subjects
with either impulsive aggressive (reviewed in Kavoussi
et al2), antisocial personality disorders,3,4 or
attempted/completed suicide.5,6 5-HIAA is the principal metabolite of serotonin and its CSF levels correlate
with 5-HIAA concentrations in frontal cortex.7,8 Genetic epidemiological studies suggest a genetic liability
for aggressive and suicidal behaviors that is independent of or additive to any associated psychiatric
disorder.9–11
Tryptophan hydroxylase (TPH) is the rate-limiting
enzyme in the biosynthetic pathway of serotonin.
Therefore, inherited variants of TPH may account for
the reduction in CSF 5-HIAA levels observed in impulsive aggression and suicidality. We previously reported
both association and linkage of a TPH intron 7 variant
(A to C transversion at position 779) (TPH
IVS7+779C⬎A) to suicidality among Finnish impulsive
offenders.12,13 Association of the TPH IVS7+779C⬎A
with suicidality was also observed in a group of 51
American Caucasians with major depression.14 Belli-
Tryptophan hydroxylase promoter variants and behavior
A Rotondo et al
vier et al15 found a significant association between the
TPH polymorphic allele IVS7+218C⬎A, which is
almost in complete disequilibrium with TPH
IVS7+779C⬎A,16 and bipolar disorder. They did not
find any statistical difference between bipolar patients
with or without a history of suicide attempts.15 However, using a linearity tendency test, an association
between the 218A allele and the violence of the suicide
attempt was observed.17 Finally, we recently reported
evidence for linkage between TPH IVS7+779C⬎A and
alcoholism in a sample of 369 sib pairs.13
Although the TPH IVS7+779C⬎A does not alter
amino acid sequence and does not appear to be directly
involved in TPH gene transcription, it could modify
gene expression by altering the processing of the TPH
pre-messenger RNA, as described in the ␤-globin
gene.18 However, no alteration in splicing was
observed when complementary DNAs from both TPH
alleles were sequenced.16 Alternatively, TPH
IVS7+779C⬎A could be a linkage disequilibrium with
an unidentified variant located in the TPH promoter
(TPH-P), elsewhere in the TPH gene or even in a
nearby gene.
In the present study we screened the TPH promoter
(2093 nucleotides) in an ethnically diverse population
of 260 subjects. Linkage disequilibrium between the
−6526A⬎G variant and the known TPH intron seven,
IVS7+779C⬎A, polymorphism was measured in three
defined populations and association to behavior
(suicidality) was studied in 320 Finns.
Materials and methods
A sample of 260 unrelated individuals including 69
Italians, 65 American Caucasians, and 126 Finnish
Caucasians, was screened for variants in the TPH-P.
Three hundred and twenty Finnish Caucasians, 237
Italians, 174 American Caucasians, and 41 Southwestern American Indians were genotyped for the TPH
−6526A⬎G variant. The behavioral sample of 320
Finns included 167 unrelated male violent offenders
and arsonists who were ordered to undergo forensic
psychiatric examination by a district court and 153
unrelated controls. Violent offenders and arsonists
were evaluated for a history of suicide attempts by
review of their medical records. They were classified
as impulsive (n = 113) or non-impulsive (n = 54) based
on the nature of their crime. Impulsive crimes were
unpremeditated and unprovoked, whereas non-impulsive crimes were clearly premeditated.12,13 Demographic and clinical data on these subjects are reported
in Table 1. The Italian population sample (n = 237)
included 84 controls, 48 obsessive-compulsive patients
(OCD), 56 patients with eating disorders (ED), and 49
subjects suffering from bipolar I disorder. The American Caucasian population sample included 46 controls, 60 OCDs, and 68 EDs. The Southwestern American Indians consisted of 41 subjects from a
population-based sample being used to investigate the
genetics of alcoholism.19 Patients and controls were
diagnosed according to DSM-III-R criteria using the
Table 1
Demographics of the behavioral sample
Characteristic
Offenders
Controls
No.
Male sex – No. (%)
Age – yr (± sd)
Impulsive (%)
Nonimpulsive (%)
SCID lifetime diagnosisa
Alcohol dependence
Alcohol abuse
Major depression
Dysthymia
Social phobia
Simple phobia
OCDb
Schizophrenia
ASPDc
Suicide attempters
167
167 (100)
32 ± 9
113 (68)
54 (32)
112
95
16
11
9
0
0
0
3
53
97
153
153 (100)
32 ± 4
0 (0)
0 (0)
130
0
0
3
1
1
1
1
0
0
0
a
Subjects who were psychiatrically interviewed.
Obsessive-compulsive disorder.
c
Antisocial personality disorder.
b
Structured Clinical Interview for DSM-III-R, patient
edition (SCID-P). All subjects signed a written consent
form. The study was approved by the National Institute
of Mental Health Intramural Research Program Institutional Review Board and the Office of Protection
from Research Risks in the USA, by the University of
Helsinki Central Hospital and Department of Psychiatry Institutional Review Boards in Finland, and by
the Ethical Committee of the University of Pisa in Italy.
Linkage disequilibrium between the TPH −6526A⬎G
polymorphism and the TPH IVS7+779C⬎A variant was
estimated in 167 Finnish offenders, 153 Finnish controls, 84 Italian controls, and 41 Southwestern American Indians.
DNA extraction
Genomic DNA was extracted from Epstein–Barr
immortalized lymphoblastoid cells using standard protocols, or, in the case of the Italian subjects, the DNA
was extracted from blood lymphocytes.
Screening for variants of the TPH promoter region
PCR Eight sets of primers (Table 2) yielded eight
overlapping fragments encompassing 2093 nucleotides
of the TPH-P region.20 Amplification was performed
using 100 ng genomic DNA, 0.2 ␮M of each primer,
250 ␮M each of dATP, dCTP, dGTP, and dTTP, 50 mM
KCl, 1.5 mM MgCl2, 0.001% gelatin, 10 mM Tris-HCl
(pH 8.3), 0.16 ␮g TaqStart antibody (Clontech, Palo
Alto, CA, USA) and 1 unit of AmpliTaq (Perkin Elmer,
Foster City, CA, USA) in 15 ␮l total volume. For singlestrand conformation polymorphism (SSCP) analysis,
1.75–2.25 ␮Ci of [␣33P] dCTP was included in the PCR
reaction. Samples were amplified for 30 cycles, each
consisting of 1 min at 94°C, 2 min at the appropriate
361
Tryptophan hydroxylase promoter variants and behavior
A Rotondo et al
362
Table 2
Primer
1F
1R
2F
2R
3F
3R
4F
4R
5F
5R
6F
6R
7F
7R
8F
8R
ART-F
ART-R
PCR primers for amplification of overlapping fragments covering the TPH-P region
Primer sequence
5⬘-CGTTTCTGGGTTCTCTATTC-3⬘
5⬘-GGACACCAAAAGAACAGAAG-3⬘
5⬘-TTGGGTAGCATGGACATTTG-3⬘
5⬘-ATGAAAAAGAAATTAATCCC-3⬘
5⬘-TTCACTCCTTTGGTTAATTC-3⬘
5⬘-AGAAGAAATAAAGGGCATCC-3⬘
5⬘-AATTTGACTTCTTCCTTTCC-3⬘
5⬘-AGTACCATCAATAATCAAGC-3⬘
5⬘-TGGCATTGAAGTAAGAGCAC-3⬘
5⬘-GTTTCATGCAGGTATTAGTG-3⬘
5⬘-GAAAAGCTGTAAAGGTCCTG-3⬘
5⬘-GTAGTCAGTCGATTAGCTCC-3⬘
5⬘-CTCAGAATTGTACACCTCAC-3⬘
5⬘-CTGACTAACACTGAACAATC-3⬘
5⬘-GGTCTCCAACAGGCGAGAAG-3⬘
5⬘-CAGTAGGTGCAGGCTGGGTC-3⬘
5⬘-AATAGCCTTCTGTTCTTTTG-3⬘
5⬘-GTGAAAGATCTCTACAATGAC-3⬘
(G)
Nucleotide
positiona
Annealing
temperature
Restriction
enzyme
PCR product
(bp)
−2093 → −2072
−1754 → −1773
−1831 → −1812
−1631 → −1652
−1704 → −1683
−1431 → −1450
−1475 → −1454
−1200 → −1219
−1262 → −1241
−984 → −1003
−1080 → 1061
−600 → −619
−752 → −731
−205 → −224
−317 → −296
+19 → +1
−1778 → −1757
−1700 → −1719
55°C
ApoI
180–161
52°C
200
55°C
274
52°C
276
54°C
280
52°C
DpnII
201–84–184
52°C
ApoI
175–87–286
65°C
PacI
150–188
50°C
Tsp45I
58–21
a
Nucleotide position upstream the TPH transcription start site.20
annealing temperature (Table 2), and 3 min at 72°C.
Final DNA elongation was at 72°C for 7 min.
Single-strand conformation polymorphism (SSCP)
analysis Amplified DNA fragments longer than 280
base pairs were digested with restriction enzymes
(Table 2). The reaction mixture consisted of 5 ␮l of PCR
product, 5 units of enzyme, and the appropriate buffer
and was incubated for 3 h. Five microliters of reaction
mixture were diluted with 10 ␮l of 95% deionized
formamide, 10 mM NaOH, 0.05% bromophenol blue,
and 0.05% xylene cylanol, and incubated for 3 min at
95°C. Five microliters of denatured DNA were loaded
per lane in an MDE gel (FMC Bioproducts, Rockland,
ME, USA) and electrophoresed for 16 h at 4°C at 6 W.
The gels were dried and autoradiographed at −70°C.
DNA sequencing PCR products were purified by agarose gel electrophoresis followed by electroelution.
DNA was directly sequenced by Circumvent Thermal
Cycle Sequencing (New England Biolabs, Boston,
MA, USA).
Genotpying
Variants TPH −7180T⬎G, TPH −6526A⬎G, and TPH
−5806G⬎T were genotyped by PCR-RFLP methods,
while TPH −7065C⬎T was genotyped by SSCP
(Table 3).
To genotype the TPH −7180T⬎G variant by PCRRFLP, primers ART-F and ART-R were employed to
create an artificial restriction site (Table 2). ART-R
introduces a G to A substitution six nucleotides downstream from the polymorphic site, enabling the detection of this site as an Tsp 451 RFLP (Table 3). TPH
IVS7+779C⬎A genotypes were determined as previously described.16,21
Electrophoretic mobility shift assay (EMSA)
Nuclear extracts from human retinoblastoma (Y79) or
epithelioid carcinoma (HeLa) were purchased from
Santa Cruz Biotechnology (Santa Cruz, CA, USA) or
Life Technologies (Gaithersburg MD, USA), respectively. Oligonucleotides encompassing TPH-P nucleotide substitutions (Figure 1) were synthesized using an
ABI 394 DNA/RNA Synthesizer (Perkin-Elmer, Foster
City, CA, USA). Complementary strands were
denatured at 95°C for 10 min in 5 mM NaCl, 1 mM
Tris-HCl, 1 mM MgCl2, 0.1 mM DTT and annealed by
cooling to 22°C. Double-stranded oligonucleotides
were purified from 10% native polyacrylamide gels,
labeled with [␥32P]-ATP using T4 polynucleotide kinase (New England Biolabs, Boston, MA, USA) and
unincorporated nucleotides removed using G-25
Sephadex columns (5⬘ to 3⬘ Inc, Boulder, CO, USA).
Radiolabeled probe (100 000 cpm) was incubated with
6 ␮g of nuclear extract in a 10 ␮l reaction containing
5 mM Tris-HCl pH 7.5, 50 mM NaCl, 0.5 mM DTT, 0.5
mM EDTA, 5% glycerol and 1 ␮g poly(dI-dC)
(Boehringer Mannheim, Indianapolis, IN, USA). After
incubation for 20 min on ice, protein-nucleic acid complexes were loaded on a 6% polyacrylamide gel (Long
Ranger, FMC, Rockland, ME, USA) prerun for 1 h at
200 V in 0.25 × TBE (22.2 mM Tris-HCl, 22.2 mM boric
acid, 0.5 mM EDTA), electrophoresed at room temperature for 1.5 h at 200 V, fixed with 20% glacial acetic
acid, 20% methanol for 10 min, dried and exposed to
film overnight at −70°C.
Tryptophan hydroxylase promoter variants and behavior
A Rotondo et al
Table 3
363
Variants identified in the TPH promoter region
Nucleotide
substitution
Nucleotide
position
Primers
Restriction
enzyme
Allele
Fragment sizes
(base pairs)
T→G
−7180
ART-F/ART-R
Tsp45I
C→T
−7065
3F/3R
SSCP detection
58 + 21
79
274
A→G
−6526
5F/5R
Sau3AI
G→T
−5806
7F/7R
MslI
G
T
T
C
G
A
T
G
280
194 + 86
401 + 147
548
Figure 1 Electrophoretic mobility shift analysis28 of nuclear protein binding to oligonucleotides representing alleles of the
four TPH-P polymorphisms: −7180T⬎G, TPH −7065C⬎T, TPH −6526A⬎G, and TPH −5806G⬎T. Double-stranded oligonucleotide probes (listed below) labeled with [␥32P]-ATP encompassing the nucleotide substitutions (arrows) were incubated for 20
min with 6 ␮g of nuclear extracts from HeLa (epithelioid carcinoma) cells as described in the text. DNA-protein complexes
were analyzed on 6% native low ionic strength polyacrylamide gels.
Tryptophan hydroxylase promoter variants and behavior
A Rotondo et al
364
Statistical analysis
Genotype and allele frequencies comparisons between
affected and unaffected individuals were carried out
using the ␹2 test and analysis of variance (ANOVA)
were performed for comparison of continuous variables (Statview, Abacus Concepts, Berkeley, CA, USA).
Haplotype estimation and linkage disequilibrium calculations were performed using the 3LOCUS.PAS program.22
Results
Variant identification in the TPH promoter region
In a sample of 260 individuals, four common polymorphisms (TPH −7180T⬎G, TPH −7065C⬎T, TPH
−6526A⬎G, and TPH −5816G⬎T (Table 3), designated
as nucleotides upstream of the translation start site20,23)
were identified in the TPH-P by SSCP analysis and
direct sequencing of PCR fragments. In the Finns (n =
126), all TPH-P variants found were in complete linkage disequilibrium resulting in two haplotypes:
G:T:G:T and T:C:A:G. In the Italians (n = 69) and American Caucasians (n = 65), all subjects showed the haplotypes G:T:G:T and T:C:A:G, except one Italian had the
G:C:A:G haplotype and one American Caucasian had
the G:C:A:G haplotype, one had T:T:G:T and one was
a T:T:G:T homozygote. No statistical differences in
genotype frequencies were observed in the three populations. Genotype and allele frequencies for the TPH
− 6526A⬎G variant are shown in Table 4. The genotypic distributions of the four polymorphisms were in
Hardy–Weinberg equilibrium in each population
examined.
Linkage disequilibrium between the TPH −6526A⬎G
and TPH IVS7+779C⬎A21 polymorphic alleles was
estimated in 167 Finnish offenders, 153 Finnish controls, 84 Italian controls, and 41 Southwestern American Indians. As shown in Table 5, significant linkage
disequilibrium (LD) between −6526A⬎G and +779C⬎A
was observed in both Finnish samples (P ⬍ 0.001) with
a normalized LD of 0.89. However, in the Italians and
in the Southwestern American Indians, the normalized
LD between the two markers was only 0.49 (P ⬍ 0.001)
and 0.21 (n.s.), respectively (Table 5).
Association of TPH promoter variants with
impulsivity and suicidality in Finnish offenders
Both the representative TPH-P variant −6526A⬎G and
the TPH intron 7 variant were associated with suicidalTable 4
ity in the Finnish offenders and the association was
stronger in the impulsive sub-group (Tables 6 and 7).
In the non-impulsive sub-group, no association was
observed with the TPH −6526A⬎G variant, while a
modest association was observed with the TPH
IVS7+779C⬎A polymorphism. When controls were
compared to offenders, impulsive or non impulsive
offenders, suicide attempters, subjects with antisocial
personality disorder or with alcohol dependence, no
statistically significant differences were found in
−6526A⬎G or IVS7+779C⬎A genotype or allele frequencies (data not shown). However, both the −6526A
and the IVS7+779C alleles had a higher frequency in
the control group as compared to the non-suicide
offenders (for −6526A: 0.43 vs 0.26, ␹2 11.24, P =
0.0008; for IVS7+779C: 0.56 vs 0.41, ␹2 8.46, P = 0.004)
and to the non-suicide impulsive offenders (for
−6526A: 0.43 vs 0.21, ␹2 12.69, P = 0.0004; for
IVS7+779C: 0.56 vs 0.35, ␹2 11.48, P = 0.0007).
The IVS7+779C⬎A and −6526A⬎G haplotypes were
evaluated in the control, offenders, impulsive
offenders, and nonimpulsive offenders groups. As
expected, a highly significant association of TPH
alleles with suicidality was found in the violent
offenders and in the impulsive violent offenders subgroups, but not in the nonimpulsive sub-group (Table
8). No statistically significant differences were
observed in haplotype frequencies between control
and patient groups (data not shown). The
−6526G/IVS7+779C haplotype was associated with
suicidality and the −6526A/IVS7+779A was associated
with an absence of this behavior. After correction for
multiple testing, the association between suicidal
behavior and TPH alleles, genotypes and haplotypes
remains highly statistically significant (data not
shown).
EMSA analysis
To identify altered protein-nucleic acid interations in
the TPH promoter regions at the variant sites, electrophoretic mobility shift assays (EMSAs) were performed. Double-stranded radiolabeled oligonucleotide
probes containing each of the allelic variants were
incubated with nuclear extracts from HeLa (epithelioid
carcinoma) or Y79 (retinoblastoma) cell-lines (Figure
1). Variation in protein binding was observed at three
of the four variant sites. An increase in binding was
observed at the TPH −7180T⬎G site while relatively
TPH −6526A⬎G genotypes in various populations
TPH −6526A⬎G
genotype
Italians
US Caucasians
Finns
American Indians
AA
0.34
0.36
0.36
0.27
(80)
(63)
(114)
(11)
AG
0.50
0.38
0.49
0.66
(117)
(66)
(156)
(27)
Values are frequencies with number of subjects in parentheses.
GG
0.17
0.26
0.16
0.07
(40)
(45)
(50)
(3)
A allele frequency
n
0.58
0.55
0.60
0.60
237
174
320
41
Tryptophan hydroxylase promoter variants and behavior
A Rotondo et al
Table 5
365
Linkage disequilibrium between the TPH IVS7+779C⬎A and −6526A⬎G loci
Population
Finnish offenders
Finnish controls
Italians
Southwestern American Indians
D⬘
D
Haplotypes
P-value
0.915
0.859
0.494
0.214
0.159
0.162
0.103
0.041
334
306
168
82
⬍0.0001
⬍0.0001
⬍0.0001
n.s.
D⬘: normalized linkage disequilibrium.
D: linkage disequilibrium.
P-value: deviation from linkage equilibrium.
Table 6
Population association between TPH IVS7+779C⬎A and suicidality
History of suicide attempts
Impulsive
Genotype
779A/779A
779A/779C
779C/779C
Total
P=
779C Allele
Frequency
P=
Nonimpulsive
All offenders
Controls
With
# %
Without
# %
With
# %
Without
# %
With
# %
Without
# %
# %
7 (9)
44 (61)
22 (30)
18 (45)
16 (40)
6 (15)
5 (21)
6 (25)
13 (54)
6 (20)
18 (60)
6 (20)
12 (12)
50 (52)
35 (36)
24 (35)
34 (48)
12 (17)
33 (22)
68 (44)
52 (34)
73
40
24
30
97
70
153
0.41
0.56
⬍0.0001
0.60
0.017
0.35
0.67
0.0003
0.0008
0.50
0.62
0.08
0.0002
P, probabilities were calculated with the ␹2 test.
Table 7
Population association between TPH −6526G⬎A and suicidality
History of suicide attempts
Impulsive
Genotype
−6526A/−6526A
−6526G/−6526A
−6526G/−6526G
Total
P=
−6526A Allele
Frequency
P=
Nonimpulsive
All offenders
Controls
With
# %
Without
# %
With
# %
Without
# %
With
# %
Without
# %
# %
18 (25)
43 (59)
12 (16)
25 (63)
13 (32)
2 (5)
9 (37)
10 (42)
5 (21)
13 (43)
14 (47)
3 (10)
27 (28)
53 (55)
17 (17)
38 (54)
27 (39)
5 (7)
49 (32)
76 (50)
28 (18)
40
24
30
97
70
153
0.26
0.43
73
0.0003
0.46
0.54
0.21
0.0002
P, probabilities were calculated with the ␹2 test.
0.42
0.0016
0.33
0.37
0.45
0.0006
Tryptophan hydroxylase promoter variants and behavior
A Rotondo et al
366
Table 8
TPH haplotypes and suicidality
History of suicide attempts
Impulsive
Haplotype
−6526A⬎G
A
A
G
G
Total haplotypes:
P=
IVS7+779C⬎A
A
C
A
C
Non impulsive
All Offenders
Control
Witha
Without
With
Without
With
Without
Without
0.39
0.15
0.01
0.45
0.64
0.15
0.01
0.20
0.33
0.25
0.00
0.42
0.45
0.21
0.05
0.29
0.37
0.18
0.01
0.44
0.55
0.18
0.03
0.24
0.41
0.16
0.03
0.40
80
48
60
194
140
302
146
0.0009
n.s.
0.0003
a
Haplotype frequency.
P, probabilities were calculated with the ␹2 test.
minor alteration in binding was observed at the TPH
−6526A⬎G and −5806G⬎T sites.
Discussion
We have identified in the TPH promoter four common
polymorphisms: −7180T⬎G, −7065C⬎T, −6526A⬎G,
and −5806G⬎T (schematically displayed in Figure 2).
These variants are located −1721, −1606, −1067 and
−347 nucleotides, respectively, upstream of the TPH
transcription start site which itself is located at −5459
nucleotides upstream of the translational start site. In
the Finns these variants are in complete linkage disequilibrium with each other resulting in two haplotypes: G:T:G:T and T:C:A:G, however two other uncommon haplotypes (G:C:A:G and T:T:G:T) were observed
in an Italian and in an American Caucasian. No polymorphisms were observed in the region of the TPH-P
spanning from the transcription initiation site to −252
nucleotides which includes a number of putative cisregulatory elements that have been shown to have a
significant effect on basal transcriptional activity.20
However, there remains the possibility that we have
missed one or more variants by using the SSCP technique as a polymorphism screening procedure, because
the average efficiency of this method is about 80%.24
To identify if the variants are likely to alter a known
transcription factor binding site, the TRANSFAC
(Transcription Factor Database) at EMBL was searched.
The TPH −7180T⬎G polymorphism alters an AP-1
binding site as well as the SRY (Sex determining factor)
binding site. The TPH −7065C⬎T and TPH −6526A⬎G
sites lie within a C/EBP binding site (CAAT enhancer
binding protein) and a c-ETS binding site, respectively,
but do not alter the core binding sequence. The TPH
−5806G⬎T polymorphism does not alter any known
DNA binding protein consensus site. EMSA analysis
was employed to empirically assess the effect of these
nucleotide substitutions on protein-nucleic acid interactions. Three of the four sites displayed altered
nuclear protein binding. A difference in protein binding was observed at the −6526A⬎G site. The significance of these observations is unknown, however it is
possible that reporter gene assays will reveal differences in the TPH-P activity between the variant alleles.
No significant differences were observed in
−6526A⬎G allele frequencies among four populations
(Table 4). This finding is similar to observations on the
intron seven variant, TPH IVS7+779C⬎A, in this and
in a previous study.16
Linkage disequilibrium between the TPH −6526A⬎G
and the IVS7+779C⬎A, located approximately 20 000
Figure 2 Schematic of known variants in the TPH gene. Listed below the diagram of the TPH gene (open boxes = exons) are
the six known variants in the TPH gene. The sequence surrounding each variant is displayed. In parentheses beneath each
TPH-P variant is the variant’s location upstream of the transcription start site. The arrow indicates the transcription start site
and the ATG indicates the translation start site.20,23
Tryptophan hydroxylase promoter variants and behavior
A Rotondo et al
nucleotides apart, differed among various populations
but was strong in the Finns. The lower normalized
linikage disequilibrium in the American Indians (0.21)
and Italians (0.49) presumably reflects differences in
population history and structure. The Finnish population is thought to have been founded in the past 4000
years, although it has been shown that the population
had a dual Asiatic and European origin.25 It remains
possible that the TPH-P and TPH intron 7 variants are
not themselves functionally significant but, rather, are
in linkage disequilibrium with a functional variant in
TPH or at a neighboring locus that could influence
behavior. Since the linkage disequilibrium between the
TPH −1066G⬎A and the IVS7+779C⬎A alleles is lower
in the other populations studied, it may be advantageous to use these populations to genetically map the
location of the functional variant producing the associations to behavior.
The TPH −6526G allele, as well as the TPH
IVS7+779C allele, was associated with suicidality in
our sample of Finnish offenders. This sample represents a subset of the sample used for previous studies.12,13 Similar to earlier findings,12,13 this association
was found to be most significant in the impulsive
offenders. The TPH-P and TPH intron 7 variants were
not associated with impulsivity, alcoholism, or antisocial personality disorder in this study. However, the
TPH intron 7 marker has previously been shown to be
linked to alcoholism and socialization score on the
Karolinska Scales of Personality in Finns.13 Interestingly, impulsive offenders who did not attempt suicide
showed significantly higher frequencies of the −6526A
and the IVS7+779A alleles compared to the controls.
Thus, the −6526A/IVS7+779A allele may have a ‘protective’ effect within impulsive subjects at high risk of
suicide or be in linkage disequilibrium with such a
polymorphism.
In conclusion, this study extends our previous findings of an association of TPH genotypes with suicidality among Finnish impulsive offenders.12,13 If neither
the TPH IVS7+779C⬎A polymorphism16 nor the TPHP variants are functional, the linkage disequilibrium
presented here suggests that these polymorphisms
could be in linkage disequilibrium with another functional variant within the TPH locus or nearby. Studies
on the association between the TPH gene and suicidality are inconsistent. An association of the TPH
IVS7+779A allele with suicidality was observed in 51
American
Caucasians
suffering
from
major
depression.14 On the contrary, Abbar et al26 found no
association between an undefined polymorphic Ava II
restriction site in the TPH locus and suicidality in a
sample of the Caucasian European population. Similarly, Bellivier et al15 did not find any significant difference in allele frequency for the TPH IVS7+218C⬎A
variant between bipolar patients of French origin with
or without a history of suicide attempts, although,
using the linearity tendency test, they observed a significant association between the +218A allele and the
violence of the suicide attempt.17 The discordance
between our results and those of the Abbar26 and
Bellivier15 groups could arise due to differences in
linkage disequilibrium relationships in different populations, as well as differences in patient sample composition or patient ascertainment.27
Acknowledgements
We thank Longina Akhtar for her excellent technical
assistance, Dr Chiara Mazzanti for her molecular genetic assistance, and Dr Raymond Peterson for his statistical genetic assistance. A Rotondo was in part supported by the IDEA Association (Institute for the
Treatment and Prevention of Depression and Anxiety),
via Statuto 8, 20121 Milan, Italy, and by the Association Fredom From Fear, 308 Seaview Avenue, Staten
Island, New York, NY 10303, USA.
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