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Dopamine beta-hydroxylase (DBH) activity and -1021CT polymorphism of DBH gene in combat-related post-traumatic stress disorder.

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American Journal of Medical Genetics Part B (Neuropsychiatric Genetics) 144B:1087– 1089 (2007)
Brief Research Communication
Dopamine Beta-Hydroxylase (DBH) Activity
and 1021C/T Polymorphism of DBH Gene in
Combat-Related Post-Traumatic Stress Disorder
Maja Mustapić,1 Nela Pivac,1 Dragica Kozarić-Kovačić,2 Martina Deželjin,1 Joseph F. Cubells,3
and Dorotea Mück-Šeler1*{
1
Division of Molecular Medicine, Rudjer Bosković Institute, Zagreb, Croatia
Department of Psychiatry, University Hospital Dubrava, Referral Centre for the Stress Related Disorders of the Ministry of Health and
Social Wellfare of the Republic of Croatia, Regional Centre for Psychotrauma, Zagreb, Croatia
3
Departments of Human Genetics, Psychiatry and Behavioral Sciences, Emory University School of Medicine, Atlanta, Georgia
2
The roles of dopamine (DA) and norepinephrine
(NE) in posttraumatic stress disorder (PTSD) are
unclear. The aim of the study was to determine
plasma dopamine beta-hydroxylase (DBH) activity and DBH-1021C/T gene polymorphism in combat veterans with (N ¼ 133) or without (N ¼ 34)
chronic PTSD. Similar frequencies in genotype or
allele distribution were found between veterans
with or without PTSD. War veterans with PTSD
had lower DBH activity, associated with the DBH1021C/T variant in DBH genes, than veterans
without PTSD. A significantly lower plasma DBH
activity was found in combat veterans with PTSD
carrying the CC genotype as compared to veterans
without PTSD carrying the corresponding genotype. Since both groups were exposed to the same
trauma, it is possible that a pre-existing trait
difference in regulation of NE function contributed to a differential vulnerability to develop
PTSD, or that the regulation of DBH expression
was different in response to trauma. The results
suggest that that genotype-controlled measurement of plasma DBH activity might be used as
a potential biological marker of the response to
trauma, and that further studies of DBH and
other loci related to DA and NA in PTSD are
warranted.
ß 2007 Wiley-Liss, Inc.
KEY WORDS:
Dopamine-beta-hydroxylase; DBH
polymorphism;
post-traumatic
stress disorder; war veterans
Please cite this article as follows: Mustapić M, Pivac N,
Kozarić-Kovačić D, Deželjin M, Cubells JF, Mück-Šeler
D. 2007. Dopamine Beta-Hydroxylase (DBH) Activity
and 1021C/T Polymorphism of DBH Gene in CombatRelated Post-Traumatic Stress Disorder. Am J Med
Genet Part B 144B:1087–1089.
{
Senior scientist.
*Correspondence to: Dorotea Mück-Šeler, B.Sc., Ph.D, Division
of Molecular Medicine, Rudjer Bosković Institute, PO Box 180,
HR-10002 Zagreb, Croatia
Received 6 November 2006; Accepted 15 February 2007
DOI 10.1002/ajmg.b.30526
ß 2007 Wiley-Liss, Inc.
Post-traumatic stress disorder (PTSD) is a complex psychiatric and polygenetic disorder that occurs in some people
exposed to extreme traumatic events [Nemeroff et al., 2006].
PTSD is associated with alterations in dopamine (DA) and
norepinephrine (NE)-mediated neurotransmission [Glover
et al., 2003; Hamner and Diamond, 1993; Southwick et al.,
1999]. Dopamine beta-hydroxylase (DBH) is a critical enzyme
in the catecholamine synthesis that converts DA to NE, and an
obvious candidate for the modification of the pathophysiology
of PTSD. Plasma DBH activity is under genetic control, with
the structural locus DBH accounting for more than 50% of the
variance in the trait. A single-nucleotide polymorphism (SNP)
in the 50 flanking region of DBH, -1021C/T (rs1611115)
accounts for 35–52% of the inter-individual variations in
plasma DBH activity [Zabetian et al., 2001]. Given this strong
genetic influence on plasma DBH activity, Cubells and
Zabetian [2004] have pointed out that appropriate interpretation of plasma DBH in behavioral studies requires controlling
for genotype at rs1611115.
To evaluate DBH in PTSD, we genotyped the DBH-1021C/T
SNP, measured plasma DBH activity, and evaluated the
association between plasma DBH activity and DBH-genotypes
in combat veterans, matched for traumatic experience, with or
without PTSD. The aim of the study was to determine plasma
DBH activity and the frequencies of alleles at 1021C/T at the
DBH gene in war veterans with or without chronic combatrelated PTSD.
The study included 167 unrelated Croatian Caucasian
medication-free subjects from the same military unit: 133
war veterans with current and chronic PTSD (mean age SD,
40.3 7.2 years), and 34 combat exposed war veterans who had
similar experiences but who did not develop PTSD (age
38.12 4.2 years). They participated in the same military
tasks and were exposed to the same combat-related trauma.
There was no significant difference (P > 0.05) in duration of
combat exposure between combat veterans with PTSD
(3.09 0.94 years) and non-PTSD veterans (2.98 0.96 years).
PTSD diagnosis and the presence of depression were evaluated
using the Structured Clinical Interview (SCID) for DSM-IV
[First et al., 2000]. Participants were recruited from December
2002 to January 2005 from the Referral Centre for the Stress
Related Disorders, Regional Centre for Psychotrauma, Department of Psychiatry, University Hospital Dubrava. Average
scores on the Clinician-Administered PTSD Scale [CAPS;
Blake et al., 1995] were 85.1 5.0 and 19.3 12.1 in veterans
with and without PTSD, respectively. Inclusion criteria:
combat veterans between 18 and 65 years old with total
Hamilton Rating Scale for Depression scores less than 18
[Hamilton, 1960]. Exclusion criteria: a positive history of
schizophrenia spectrum disorders, bipolar disorder, history of
cognitive dysfunction or mental retardation, history of alcohol
1088
Mustapić et al.
of other substance abuse or dependance disorders within
3 months, significant risk of violence or suicide, serious
concomitant medical condition, clinically significant abnormalities in electrocardiogram or laboratory findings, including
positive urine screen for illicit drugs. All participants were
medication-free and received no psychotropic medications,
beta-blockers or other drugs for at least 2 weeks before blood
sampling, except for diazepam (10 mg) for night-time sedation.
Written informed consent was obtained from all participants, under procedures approved by Ethics committee of the
University Hospital Dubrava, Zagreb, Croatia.
Plasma was separated from the blood samples containing
8 ml of whole blood and 2 ml of acid-citrate-dextrose anticoagulant, by centrifugation at 5,000g for 10 min, and stored
at 208C until assayed. DBH activity was determined in
duplicate by the method of Nagatsu and Udenfriend [Nagatsu
and Udenfriend, 1972], using tyramine as a substrate. The
product octopamine was oxidized to p-hydroxybenzaldehide
and measured photometrically at 330 nm.
Genomic DNA was extracted from whole blood using a
salting out procedure. DNA was amplified using primers for
polymerase chain reaction (PCR); sense primer: 50 –GGA GGG
ACA GCT TCT AGT CC-30 ; antisense primer: 50 –CAC CTC
TCC CTC CTG TCC TCT CGC-30 (5). PCR reaction conditions
and reagents (total volume 15 ml) followed the protocol
described by Köhnke et al. [2002]. Taq polymerase used was
Taq DNA GOLD (Applied Biosystems, Foster City, CA). PCR
reaction aliquots of 10 ml were digested for 3–4 hr with 3 units
of HhaI (Takara, Japan), in a final volume of 20 ml. The digested
products were separated on 4% agarose gels and visualized
with ethidium bromide under UV light. For the estimation of
fragment sizes a 50 bp ladder DNA (Takara, Japan) was used.
Plasma DBH activity and DBH genotype and allele frequencies
were determined in all subjects.
Plasma DBH activity was non-normally distributed, and
therefore the results were square-root transformed [Zabetian
et al., 2001], and evaluated using one-way and two-way
analysis of variance (ANOVA). The chi-square (w2) test was
used to test differences in genotype and allele frequencies of
DBH 1021C/T polymorphism among war veterans with or
without PTSD. The statistical packages used were Statistica 6.
Deviations from Hardy–Weinberg equilibrium were assessed.
Plasma DBH activity was significantly lower (F(1,165) ¼
41.31, P < 0.001) in war veterans with PTSD than in veterans
without PTSD (Fig. 1).
Fig. 1. Plasma DBH Activity in war veterans with and without PTSD.
Each column represents mean SD. The number of subjects is given in
parenthesis. *P < 0.05 versus war veterans without PTSD.
Among 167 subjects CC, CT, TT genotype was found in 62%,
34%, and 7% subjects, respectively. The T allele frequency was
0.22. There was no significant (P > 0.05) deviation from the
Hardy –Weinberg distribution for any group.
DBH genotype (w2 ¼ 2.30; df ¼ 2; P ¼ 0.32) and allele
(w2 ¼ 2.01; df ¼ 1; P ¼ 0.156) frequencies were similarly distributed between the two groups of war veterans. CC genotype
was found in 59%, TT genotype in 5%, and CT genotype in 36%
of veterans with PTSD. Within veterans without PTSD, 74%
carried CC, 3% TT, and 23% CT genotype. C allele carriers were
77%, and 85%, T allele carriers were 23% and 15% in veterans
with and without PTSD, respectively.
A two-way ANOVA revealed a significant separate effect of
diagnosis (F ¼ 5.92, df ¼ 1, P ¼ 0.016) and genotype (F ¼ 31.0,
df ¼ 2, P ¼ 0.0001) on plasma DBH activity in war veterans,
and a trend for an interaction between diagnosis and genotype
(F ¼ 2.83, df ¼ 2, P ¼ 0.062). The interaction was followed up by
comparing veterans with or without PTSD within each
genotype using one-way ANOVAs (Fig. 2). Within the CC
genotype, veterans with PTSD had lower plasma DBH activity
than veterans without PTSD (F(1,102) ¼ 48.82, P < 0.001).
Participants carrying CT or TT genotypes showed no significant difference in plasma DBH activity.
The present work shows for the first time a lower DBH
activity in war veterans with PTSD compared to war veterans
without PTSD. The results suggest that combat trauma
survivors could be divided in two groups including subjects
adaptable to trauma, who exhibited higher plasma DBH
activity post-trauma and those less resilient and more
vulnerable to develop PTSD, who exhibit lower plasma DBH
activity post-trauma. Since both groups were exposed to the
same trauma, it is possible that a pre-existing trait difference
in NE function contributed to a differential vulnerability to
develop PTSD. However, given the strong genetic influence on
plasma DBH activity [Weinshilboum, 1978], and the lack of
evidence in any other study of separate enzyme-activity groups
within 1021C > T genotype groups [reviewed by Cubells and
Zabetian, 2004], a more likely interpretation is that group
differences in plasma DBH activity within genotype groups
represent a differential response of the noradrenergic system
to exposure to combat trauma.
A similar finding, of genotype-independent differences
in plasma DBH activity, has been reported in psychotic
Fig. 2. Plasma DBH activity and DBH genotypes of the DBH-1021C/T
SNP in war veterans with and without PTSD. Each column represents
mean SD. The number of subjects is given in parenthesis. *P < 0.05 versus
war veterans without PTSD carrying CC genotype.
DBH in Posttraumatic Stress Disorder
depression [Cubells et al., 2002]. As in PTSD, psychotic
depression is associated with dysregulation of the hypothalamic-pituitary axis [Nelson and Davis 1997]. One possible
explanation for a trauma-dependent alteration in plasma DBH
activity within genotype group is that chronic dysregulation of
the HPA axis results in altered expression of DBH protein
[Cubells and Zabetian, 2004].
The role of DBH in the etiology of PTSD is not clear, but
altered DBH expression could in turn affect DA [Sher et al.,
2005] or NE [Southwick et al., 1999]. Although we did not
measure plasma DA and NE levels, higher plasma [Hamner
and Diamond, 1993] and urine [Glover et al., 2003] DA levels,
and equal [Southwick et al., 1999], or higher plasma NE levels
[Yehuda et al., 1998], or similar 24-hr urine NE and 3-metoxy4-hydroxyphenyglycol excretion [Mellman et al., 1995] were
found in PTSD compared to controls subjects.
Lower plasma DBH activity has been reported in paranoid
schizophrenia, unipolar geriatric delusional depression, and
unipolar depression with psychotic features [Cubells and
Zabetian, 2004]. However, a study of PTSD subjects found
higher plasma DBH activity in a subset of psychotic PTSD
subjects [Hamner and Gold, 1998]. The difference between this
study and our data might be explained by the fact that our
study included larger groups of war veterans, and that plasma
DBH activity was higher in subjects carrying CC genotype of
the DBH 1021C/T polymorphism, which was not assessed in
the Hamner and Gould [1998] study. Aside from different
genotypes, lower plasma DBH activity might be affected by
previous medication, as exposure to neuroleptics decreases
plasma DBH activity [Markianos et al., 1976]. Since our
subjects were medication-free, the possible effect of neuroleptics on plasma DBH activity was excluded. Lower plasma DBH
activity might also be due to a change in the NE neuronal
function [Southwick et al., 1999], thus causing a change in the
level of DA/NE conversion, followed by negative feedback on
plasma DBH activity and NE synthesis [Cubells and Zabetian,
2004].
In support of previous findings [Zabetian et al., 2001], we
found that DBH 1021C/T strongly associates with activity of
plasma DBH in a Croatian sample, with CC, CT, and TT
genotypes associated with higher, intermediate, and lower
plasma DBH activity, suggesting co-dominant inheritance.
Plasma DBH activity was lower in war veterans with PTSD
carrying CC genotype compared to non-PTSD veterans of the
same genotype. A similar association between plasma DBH
activity and CC genotype of the DBH-1021C/T SNP has been
reported in alcoholic subjects [Köhnke et al., 2002].
The major limitation of the study is the small number of
subjects with non-PTSD, due to the difficulty of finding combat
veterans without PTSD. However, in spite of this limitation the
findings appear consistent with prior literature.
In conclusion, the results of the present study show
alterations in plasma DBH activity and differences in war
veterans with PTSD carrying the CC genotype, indicating that
NE-mediated mechanisms are altered in PTSD. Our results
suggest that genotype-controlled measurement of plasma
DBH activity might be used as a potential biological marker
of the response to trauma.
ACKNOWLEDGMENTS
This study was supported by the Croatian Ministry of
Science, (Grant No. 0098088 – DMS). Additional support was
provided by National Institutes of Health (Grant No. K02 DA
015766 – JFC). We thank Tanja Jovanovic, Ph.D. (Emory
University, Atlanta GA, USA) for the helpful comments.
1089
Thanks are due to the staff of the Department of Psychiatry,
Referral Centre for the Stress-related Disorders, Regional
Centre for Psychotrauma, University Hospital Dubrava,
Zagreb, Croatia.
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