Neuron, Vol. 45, 11–16, January 6, 2005, Copyright ©2005 by Elsevier Inc.DOI 10.1016/j.neuron.2004.12.014
ReportLoss-of-Function Mutation in
Tryptophan Hydroxylase-2 Identified
in Unipolar Major Depression
2004). Numerous studies have suggested associations
between various neuropsychiatric disorders and genes
that modulate central serotonergic neurotransmission,
such as the 5-HT transporter (SERT, 5-HTT) (Lesch et
al., 1996; Caspi et al., 2003; Murphy et al., 2003), 5-HT
receptors (Lucki, 1998; Arango et al., 2003; Bonasera
and Tecott, 2000; Gordon and Hen, 2004; Malhotra et
al., 2004), and monoamine oxidases (Shih et al., 1999),
as well as the rate-limiting enzymes in 5-HT synthesis,
tryptophan hydroxylases (TPH1 and TPH2) (Arango et
al., 2003; Harvey et al., 2004).
TPH1 and TPH2 belong to the superfamily of aromatic
amino acid hydroxylases, which also includes tyrosine
hydroxylase (TH) and phenylalanine hydroxylase (PAH).
These four enzymes share considerable structural simi-
controls most of peripheral 5-HT synthesis (Cote et al.,
2003; Walther et al., 2003), TPH2 was recently discov-
ered and found to be neuronal specific and predomi-
nantly expressedin brainserotonergic neuronsoriginat-
ing from raphe nuclei (Walther et al., 2003; Patel et
al., 2004). We have previously identified a functional
(C1473G) single nucleotide polymorphism (SNP) which
results in the replacement of proline with arginine at
position 447 in mouse Tph2 (Zhang et al., 2004). Expres-
sion of mutant Tph2 (P447R) in PC12 cells revealed an
?55% decrease in 5-HT levels as compared to wild-type
Tph2. Moreover BALB/cJ and DBA/2 inbred mice car-
rying the homozygous mutant (1473G) alleles showed
50%–70% reduction in the rate of synthesis of cortical
and striatal 5-HT accompanied by ?40% reduction in
5-HT tissue content when compared to C57Bl/6 and
129X1/SvJ inbred mice carrying homozygous wild-type
(1473C) alleles. Interestingly, these strains of mice dis-
play significantly different behaviors and responses to
antidepressants (Lucki et al., 2001). These observations
provided direct evidence for the fundamental role of
Tph2 in brain 5-HT synthesis (Zhang et al., 2004) and
raised the interesting possibility that similar mutations
in human TPH2 (hTPH2) may affect brain 5-HT homeo-
stasis in certain neuropsychiatric conditions.
Here we report the identification of a functional
(G1463A) SNP in hTPH2, which replaces a highly con-
in activity when expressed in cell culture systems. Fur-
thermore, we identified nine subjects carrying this func-
tional SNP in a cohort of 87 unipolar major depression
patients. These data provide a potential molecular
Xiaodong Zhang,1Raul R. Gainetdinov,1
Jean-Martin Beaulieu,1Tatyana D. Sotnikova,1
Lauranell H. Burch,2Redford B. Williams,3,4
David A. Schwartz,2K. Ranga R. Krishnan,4
and Marc G. Caron1,3,*
1Department of Cell Biology
Center for Models of Human Disease
Institute for Genome Sciences and Policy
2Department of Pulmonary, Allergy,
and Critical Care Medicine
3Department of Medicine
4Department of Psychiatry and Behavioral Sciences
Duke University Medical Center
Durham, North Carolina 27710
Dysregulation of central serotonin neurotransmission
has been widely suspected as an important contribu-
tor to major depression. Here, we identify a (G1463A)
single nucleotide polymorphism (SNP) in the rate-lim-
iting enzyme of neuronal serotonin synthesis, human
in hTPH2 replaces the highly conserved Arg441 with
production when hTPH2 is expressed in PC12 cells.
Strikingly, SNP analysis in a cohort of 87 patients with
unipolar major depression revealed that nine patients
carried the mutant (1463A) allele, while among 219
controls, three subjects carried this mutation. In addi-
tion, this functional SNP was not found in a cohort of
60 bipolar disorder patients. Identification of a loss-
of-function mutation in hTPH2 suggests that defect in
brain serotonin synthesis may represent an important
risk factor for unipolar major depression.
The neurotransmitter serotonin (5-hydroxytryptamine
[5-HT]) has been implicated in a variety of physiological
functions in both peripheral and central nervous sys-
tems (CNS) (Lucki, 1998; Veenstra-VanderWeele et al.,
2000; Malhotra et al., 2004). Many neuropsychiatric
disorders, such as depression (Lesch, 2004; Malhotra
et al., 2004), schizophrenia (Veenstra-VanderWeele et
al., 2000), autism (Veenstra-VanderWeele and Cook,
2004), aggression and suicidal behavior (Arango et al.,
2003), and attention-deficit/hyperactivity disorder (ADHD)
(Gainetdinov et al., 1999; Quist and Kennedy, 2001), are
considered to be related to dysfunction in serotonergic
neurotransmission inthe CNS.Therefore, thebrain 5-HT
system is a major target for tricyclic antidepressants,
selective serotonin reuptake inhibitors (SSRIs), mono-
(Lucki, 1998; Gainetdinov and Caron, 2003; Blier and
Abbott, 2001; Gordon and Hen, 2004; Malhotra et al.,
In order to explore whether functional SNP(s) could be
identified in hTPH2, we screened 11 exons of hTPH2 by
sequence analysis in 48 genomic DNA samples ran-
domly selected from a cohort of ?300 individuals from
a study of psychosocial and behavioral risk in lower
Figure 1. (G1463A) SNP in hTPH2 and Partial
Sequence Alignment of TH, PAH, TPH1,
(A) The (G1463A) polymorphism in hTPH2.
The G/A polymorphism is highlighted with an
arrowhead. Nucleotide numbers are shown
as the site of polymorphism. (B) Sequence
alignment of the C-terminal regions of tyro-
sine hydroxylase (TH), phenylalanine hydrox-
ylase (PAH), TPH1 and TPH2 in human (h),
mouse (m), and rat (r). The highly conserved
arginine residues are highlighted with an
arrowhead. Numbers indicate positions of
social-economical characteristic groups (Williams et al.,
2003). Among these samples, one novel (G1463A) SNP
in the coding region of hTPH2 was identified, which
replaced Arg441 (CGT) with His (CAT) (Figure 1A). Se-
quence alignment among TH, PAH, TPH1, and TPH2 in
human, mouse, and rat revealed that Arg441 is highly
conserved among members of this family (Figure 1B).
Interestingly, an R408W mutation at the corresponding
ity and leads to phenylketonuria (PKU), the most severe
form of hyperphenylalaninemia. Furthermore, this R408W
mutation has been identified in ?10% of the PKU pa-
tients, representing the most prevalent pathogenic mu-
tation in PAH (Scriver et al., 2003).
To analyze the functional consequence of the R441H
mutation in hTPH2 on 5-HT synthesis, we transfected
either hemagglutinin (HA)-tagged wild-type (wt) or
R441H mutant hTPH2 into pheochromocytoma (PC12)
cells and measured 5-HT levels as described (Xu et al.,
2000; Zhang et al., 2004). Previously, we established a
direct correlation between 5-HT levels measured in
PC12 cells transfected with Tph2 genetic variants and
the rate of 5-HT synthesis in the brains of inbred mice
carrying these Tph2 variants (Zhang et al., 2004). While
here we did not measure TPH2 enzyme activity directly,
we found that 5-HT levels in PC12 cells expressing the
R441H mutant were ?80% lower as compared to PC12
cells expressing wt hTPH2 (Figure 2A), indicating a se-
vere loss of function in the ability of the mutant enzyme
to synthesize 5-HT. Because humans may carry hetero-
zygous (1463G/1463A) or homozygous (1463A/1463A)
ity of this family of enzymes (Yang and Kaufman, 1994;
Fitzpatrick, 1999), we tested whether the R441H mutant
could exert a dominant-negative effect and/or affect
in PC12 cells transfected with plasmids encoding wt
hTPH2, R441H mutant hTPH2, or the combination of
both plasmids. These experiments revealed an additive,
not dominant-negative, effect of the R441H mutant on
precipitation studies in PC12 cells expressing wt-wt,
wt-R441H, or R441H-R441H hTPH2 tagged with either
FLAG or HA epitopes. As shown in Figure 2C, the R441H
mutant hTPH2 was able to form homo- (R441H-R441H)
or hetero- (wt-R441H) oligomers to a similar extent as
compared to wt hTPH2, suggesting that the R441H mu-
tation does not affect dimerization of the subunits. In
fact, the equivalent R408W mutation in PAH has been
found to affect protein folding, causing a complete loss
of protein expression and enzymatic activity as com-
pared to wtPAH (Pey et al., 2003).However, the arginine
to histidine mutation in hTPH2 did not markedly affect
hTPH2 protein expression, at least when expressed in
PC12 cells (Figure 2A, lower panel), indicating that it
ation of function than the mutant PAH.
Figure 2. Biochemical Properties of hTPH2 (R441H) Mutant
(A) 5-HT levels in PC12 cells expressing mutant R441H hTPH2 were
lower than those in PC12 cells expressing wt hTPH2 (n ? 8 experi-
ments), whereas no differences in dopamine levels were observed.
5-HT levels were normalized to the respective hTPH2 expression
levels (mutant enzyme expression was ?75% of wt levels). (B) Addi-
tive, not dominant-negative, effect of R441H hTPH2. 5-HT levels
were measured in PC12 cells transfected with plasmids expressing
wt (10 ?g), wt (5 ?g) ? R441H (5 ?g), or R441H (10 ?g) (n ? 4
experiments). (C) Immunoprecipitation of hTPH2. PC12 cells trans-
fected with wt or mutant R441H hTPH2 were subjected to immuno-
precipitation (IP) with anti(?)-FLAGantibody and immunoblotted (IB)
with anti(?)-HA antibody conjugated to horse radish peroxidase
(HRP). (All data are presented as means ? SEMs. Statistical signifi-
cance of all data presented is analyzed by Student’s t test: ***p ?
TPH2 Mutation in Depression
seven of them had a family history of mental illness or
drug and alcohol abuse, six had suicidal ideation or
attempt, four had generalized anxiety symptoms, and
interestingly, seven patients exhibited lack of respon-
siveness to SSRI, while the other two patients were
responsive only to highest doses of SSRI (Table 1). The
three control subjects carrying the 1463A allele were
not diagnosed as having unipolar major depression ac-
cording to the criteria of the Diagnostic and Statistical
clinical symptoms of comorbid conditions. One of the
three control subjects carrying homozygous mutant
the other two carrying heterozygous (1463G/1463A) al-
leles had mild depression and a family history of mental
illness or drug and alcohol abuse (Table 1), suggesting
a potentially higher susceptibility for certain neuropsy-
chiatric disorders in the presence of the mutant (1463A)
Figure 3. Allele-Specific Genotyping of (G1463A) SNP in hTPH2
(A) The (1463G) or (1463A) allele in hTPH2. Positive control primers
and allele-specific primers for genotyping are shown as black and
mic DNA carrying either (1463G) or (1463A) allele. PCR products for
positive control (492 bp) and allele-specific products (294 bp) are
indicated with arrowheads.
In this study, we report the identification of a mutation
in human TPH2 that results in ?80% loss of function in
the activity of the enzyme. Moreover, nine subjects from
a cohort of 87 unipolar major depression patients were
found to carry the mutant (1463A) allele. These findings
suggest that deficiency in brain 5-HT synthesis may be
an important risk factor for unipolar major depression.
We previously characterized a functional (C1473G)
SNP in mouse Tph2 that exhibited an ?55% reduction
in activity when expressed in PC12 cells and an ?50%
decrease in the rate of brain 5-HT synthesis and tissue
content in mice carrying the mutant allele (Zhang et al.,
2004). The highly conserved Pro residue corresponding
to this functional (C1473G) SNP in mice is Pro449 in
SNP (Arg441) reported in the present study, suggesting
that this region of hTPH2 is critical for enzyme function.
Importantly, when the R441H mutant hTPH2 was ex-
pressed in PC12 cells, 5-HT levels were reduced by
?80% as compared to wt hTPH2, indicating a more
severe loss-of-function phenotype as compared to the
mouse Tph2 (P447R) mutant. Therefore, this human
R441H mutation in hTPH2 may exert an even more pro-
found effect on brain 5-HT homeostasis.
Unipolar major depression is a brain disorder with
2%–19% population prevalence and 40%–70% herita-
tors (Lesch, 2004). Previous linkage studies have pro-
vided evidence that there is a sex-specific disposition
locus for major depression on chromosome 12q22–
ity (Abkevich et al., 2003). Human TPH2 is located on
chromosome 12q21.1 and, as we report here, a severe
loss-of-function R441H mutation in hTPH2 has been
identified in 9 out of 87 unipolar major depression pa-
tients tested. Our results that 78 unipolar major depres-
sion patients in this study carry wt (1463G) alleles are
not surprising, because depression is heterogeneous in
origin and genes other than hTPH2 or other functional
SNP(s) in hTPH2 may also contribute to vulnerability to
Based on the studies in PC12 cells, we then searched
for this functional (1463A) allele in human populations
with 5-HT-related psychiatric disorders. First, we estab-
lished a PCR-based genotyping method using a modi-
fied amplification refractory mutation system (ARMS)-
PCR (Ye et al., 2001) with one allele-specific primer and
two positive control primers flanking the site of the
(G1463A) SNP (Figure 3A) (Zhang et al., 2004). As shown
in Figure 3B, only G-allele-specific primers gave rise to
an allele-specific PCR product (294 bp) in a genomic
DNA sample carrying homozygous wt 1463G/1463G al-
leles, while both G- and A-allele-specific PCR products
were detected in a genomic DNA sample carrying het-
erozygous 1463G/1463A alleles. To further validate this
allele-specific PCR genotyping approach, we amplified
genomic DNA using the two positive control primers
mentioned above to clone the PCR fragment (492 bp)
into a cloning vector and then confirmed by sequence
analysis. Cloning vectors carrying either the (1463G) or
(1463A) allele were then used as templates to confirm
the specificity of the genotyping method (Figure 3B).
for the (G1463A) SNP in hTPH2. Using this approach, we
identified nine unipolar major depression patients and
three control subjects who carried the mutant (1463A)
allele. Nocarriers ofthe mutantallele werefound among
bipolar disorder patients (Table 1). The frequency of
the mutant (1463A) allele in unipolar major depression
patients versus control subjects was statistically signifi-
cant (?2? 13.31, p ? 0.001). No statistical difference in
the frequency of this mutant allele was present between
bipolar disorder patients and control subjects. Geno-
types of samples identified carrying the (1463A) allele
and additional samples from the control subjects (n ?
24) were then confirmed by sequence analysis of Exon
XI of hTPH2. Among the nine unipolar major depression
patients identified, six patients carried heterozygous
(1463G/1463A) alleles and three patients carried homo-
zygous (1463A/1463A) alleles (Table 1). Furthermore,
Table 1. Summary of Subjects Carrying Functional (G1463A) SNP in hTPH2
ResponsePatient I.D.SexAge Allele SuicidalityAnxietyNote
n ? 87 1202
Sertraline 200 mg
Sertraline 100 mg
n ? 2191174
Family history: family history for mental illness, drug, and alcohol abuse. Suicidality: suicidal ideation or attempt. Anxiety: three or more anxiety
symptoms. All patients listed above were Caucasians, except subject 1541, who was African American. The mutant (1463A) allele was not
found in a cohort of 60 bipolar disorder patients.
unipolar major depression. In fact, emerging evidence
indicates that genetic variance plays a critical role in
different clinical responses to psychotropic drugs (Mal-
hotra et al., 2004). For example, a growing number of
studies have suggested a relationship, although with
conflicting results, between a polymorphism in the pro-
moter region of SERT (Lesch et al., 1996) and efficacy
of SSRI to treat depression (Malhotra et al., 2004). In-
triguingly, in our study, reduced responsiveness to SSRI
in unipolar major depression patients was found in sub-
jects carrying the functional (G1463A) SNP in hTPH2,
suggesting a role of brain 5-HT synthesis in the efficacy
of SSRI treatment. It will be also important in future
studies to examine whether loss-of-function mutations
in hTPH2 may play a role in the paradoxical adverse
effects in response to SSRI treatment, such as suicid-
ality (March et al., 2004) and SSRI-exacerbated mania
and psychosis (Malhotra et al., 2004).
The three control subjects carrying the (1463A) allele
in hTPH2 were not included in the cohort of unipolar
major depression patients based on criteria of DSM-IV
diagnosis. However, the fact that these subjects had
generalized anxiety symptoms or mild depression and
a family history of mental illness or drug and alcohol
abuse further underscores the complexity and polyge-
netic nature of neuropsychiatric disorders. It is possible
that functional SNPs in hTPH2, including this (G1463A)
SNP, may be present as an important predisposing fac-
tor in the pathophysiology of several neuropsychiatric
disorders. It is also noteworthy that the functional
(G1463A) SNP was not found in a small cohort of bipolar
disorderpatients inthisstudy. Arecent geneticassocia-
tion study using a bipolar disorder French-Canadian
pedigree has identified SNPs in noncoding regions of
hTPH2, suggesting a potential role of hTPH2 in bipolar
disorder (Harvey et al., 2004). Therefore, further studies
will be needed to elucidate the relationship between
hTPH2 function and bipolar disorder.
in human TPH2, it is likely that additional functional
SNP(s) could be found in hTPH2. In support of this idea,
numerous studies have shown that there are more than
400 mutations in the closely related enzyme PAH that
cause various degrees of hyperphenylalaninemia, and
over 300 mutations identified are missense mutations
in the coding regions (Scriver et al., 2003). In fact, we
of hTPH2 that are currently under investigation (data
In conclusion, our present and previous biochemical
studies in human and mice (Zhang et al., 2004) provide
a potential molecular mechanism underlying dysfunc-
tion in 5-HT neurotransmission in the brain. Clearly, fur-
ther large-scale genetic studies are needed to confirm
tance and penetrance of this functional (G1463A) SNP
in hTPH2 in unipolar major depression. Furthermore,
detailed kinetic and biochemical characterization of the
mutant enzyme as well as direct measurements of brain
5-HT status in affected subjects will ultimately be
needed. It will also be of interest to test how this muta-
tion may interact with other genetic alterations pre-
viously associated with depression. Finally, it will be
mutations of hTPH2 in other neuropsychiatric condi-
tions, such as generalized anxiety disorder, schizophre-
nia, suicidality, autism, ADHD, and drug abuse.
to rule out neuropsychiatric disorders. They were recruited from the
community for participating in a study of psychosocial and behav-
ioral risk in lower social-economical characteristic groups (Williams
etal., 2003).Subjectsinvestigated indetailin thisstudy werepartici-
pants in the NIMH sponsored Conte Center for the Neuroscience
of Depression at Duke University. Subjects gave informed consent
prior to entry into the study. The study was approved by the Institu-
tional Review Board. The interview procedures used a structured
view uses the Diagnostic Interview Schedule (DIS) (Bosworth et al.,
2002). At baseline enrollment, 87 unrelated subjects had a DSM-IV
diagnosis of unipolar major depression and were 60 years of age
or older; they did not have to be currently depressed to participate
in this study. These subjects were not diagnosed with any other
TPH2 Mutation in Depression
psychiatric disorders. Sixty additional subjects had a DSM-IV diag-
nosis of bipolar disorder. The control group consisted of 219 sub-
jects without any reported personal history of mental illness, includ-
ing unipolar major depression, bipolar disorder, or any other major
neuropsychiatric disorders or substance abuse. Among the total
366 subjects tested in this study, 331 were Caucasians, 28 were
African Americans, 6 were Asians, 1 was Hispanic, and 41% were
male subjects. Blood was collected from these subjects, and DNA
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Primers for sequencing the 11 exons of the human TPH2 gene were
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Genotyping and PCR Conditions
ARMS-PCR genotyping was performed on human genomic DNA
samples and cloning vectors carrying either the (1463G) or (1463A)
allele. The PCR reactions were carried out with primers for positive
control (hOuter/Forward [5?-ATGTGTGAAAGCCTTTGACCCAAAG
ACA] and hOuter/Reverse [5?-TGCGTTATATGACATTGACTGAACT
GCT]) plus either G-allele- (5?-TAGGGATTGAAGTATACTGAGAAGG
cific primers. The PCR conditions were as follows: 1 cycle (5 min
at 94?C) and 40 cycles (30 s at 94?C, 30 s at 63?C, 30 s at 72?C)
using Taq DNA polymerase (Fisher). Genotyping and sequencing
experiments were carried out with investigators blinded to the clini-
cal history of patients.
To determine dopamine and 5-HT levels in PC12 cells, five million
cells were homogenized in 200 ?l 0.1 M HClO4, centrifuged, and
filtered. Supernatants were then analyzed by HPLC using electrical
chemical detection (Xu et al., 2000; Zhang et al., 2004).
GenBank Accession Numbers
TPH1 (human, NM_004179; mouse, NM_009414; rat, P09810), TPH2
(human, NM_173353; mouse, NM_173391; rat, NM_173839), TH (hu-
man, NM_000360; mouse, NM_009377; rat, NM_012740), PAH (hu-
man, NM_000277; mouse, NM_008777; rat, NM_012619).
Supported byNIH grants no. MH60451,P01HL36587, K05MH79482,
M01RR30. J.-M.B. is supported by the Human Frontiers Science
Program and the Canadian Institute of Health Research (CIHR). We
for providing DNA samples, and all subjects for participating in
Received: November 16, 2004
Revised: November 29, 2004
Accepted: December 7, 2004
Published online: December 9, 2004
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