Download Serotonin transporter gene (5-HTTLPR) is not associated

Molecular Psychiatry (1999) 4, 280–283
 1999 Stockton Press All rights reserved 1359–4184/99 $12.00
ORIGINAL RESEARCH ARTICLE
Serotonin transporter gene
(5-HTTLPR) is not
associated with depressive
symptomatology in mood
disorders
A Serretti, C Cusin, E Lattuada, D Di Bella,
M Catalano and E Smeraldi
Instituto Scientifico Ospedale San Raffaele, Department of
Neuropsychiatric Sciences, University of Milan School of
Medicine, Italy
Keywords: serotonin; polymorphism; bipolar
depressive disorder; phenotype; anxiety
disorder;
Disturbances of the serotoninergic neutrotransmitter
system have been implicated in the pathogenesis of
mood disorders. A functional polymorphism in the
upstream regulatory region of the serotonin transporter
gene (5-HTTLPR) has been recently reported to be associated with both unipolar and bipolar disorder. In this
study, we investigated the possibility that the 5-HTTLPR
might be associated with depressive symptomatology in
a sample of mood disorder subjects. One hundred and
thirty-two psychiatric inpatients affected by major
depressive (n = 67) and bipolar (n = 65) disorder (DSMIV) were assessed at admission by the Hamilton
Depression Rating Scale (HAMD-21, divided into Core,
Sleep, Activity, Psychic anxiety, Somatic anxiety and
Delusion clusters) and were typed using PCR techniques. The only prior treatment permitted was low dose
benzodiazepines (⬍5 mg diazepam or equivalent); no
prior (⬍2 weeks) antidepressant or neuroleptic treatment was allowed. 5-HTTLPR variants were not associated with total depressive symptomatology as measured
by HAMD. The short 5-HTTLPR variant was marginally
associated with higher psychic anxiety scores (F = 7.11,
d.f. = 1,262, P = 0.008). The association was stronger
among
bipolars
and
early
onset
subjects.
5-HTTLPR variants were not associated with the remaining symptom clusters. The upstream regulatory region
of the serotonin transporter gene has not, therefore, a
major influence on the depressive symptomatology in
mood disorder subjects.
Disturbances of the serotoninergic neurotransmitter
system have been implicated in the pathogenesis of
mood disorders.1,2 The serotonin transporter is the
major determinant of serotonin inactivation following
release at synapses and it is the site of action of SSRI
antidepressants. Therefore, the gene coding for the
serotonin transporter has been proposed as a candidate
for a possible involvement in the pathogenesis of mood
disorders, but the primary structure of the serotonin
transporter gene was not involved in the pathogenesis
of mood disorders.3 Recently, a polymorphic region
containing a 17-bp VNTR in the second intron was
found to be associated with unipolar disorder4 and a
functional polymorphism in the upstream regulatory
region of the serotonin transporter gene has been
associated with both unipolar and bipolar disorder,5
as well as with anxiety-related traits in normals.6 This
last is a deletion/insertion polymorphism (5-HTTLPR)
located at the 5′-flanking regulatory region of serotonin
transporter gene on chromosome 17q11.2. It consists of
a 44-bp insertion or deletion involving repeat elements
6–8 (from bp −1212 to bp −1255).6 Heils et al7 found
that the basal activity of the long 5-HTTLPR (l) variant
was more than twice that of the short 5-HTTLPR (s)
form of the 5-HTTLPR. In particular, 5-HTTLPR l form
produced steady-state concentrations of 5-HTT transporter mRNA that were 1.4–1.7 times those in cells
containing one or two copies of the 5-HTTLPR s variant
and bound 30–40% more of a labeled marker. Uptake
of labeled serotonin in cells homozygous for the
5-HTTLPR l form of the promoter polymorphism was
1.9–2.2 times that in cells carrying one or two endogenous copies of the 5-HTTLPR s variant.6 Taken together,
those studies suggest that serotonin transporter gene
transcription is modulated by variants of the 5HTTLPR, with s allele corresponding to low serotonin
uptake activity. Moreover, the data associated with the
ss and ls genotypes were similar, whereas both differed
from the ll genotype, suggesting that the polymorphism
has more of a dominant-recessive than a codominantadditive effect.
We previously analyzed 5-HTTLPR, testing a possible association with the symptomatology of major
psychoses.8–10 No influence of 5-HTTLPR variants on
psychopathology as defined by the Operational Criteria
for Psychotic Illness (OPCRIT)11 checklist has been
detected. However symptoms other than the ones
included in the OPCRIT checklist, such as psychic anxiety, somatic anxiety and hypochondria was not analyzed. Those aspects are of particular relevance as antidepressant drugs with serotonin-modulating activity
are effective on anxiety symptoms.
The aim of the present study was to investigate a
possible influence of 5-HTTLPR polymorphism on the
symptomatology of major depression defined using the
Hamilton rating scale for depression (HAMD)12 in a
sample of mood disorder subjects.
5-HTTLPR variants were not associated with the
total depressive symptomatology (Table 1). Both s
allele and s-containing genotypes were marginally
associated with higher scores on the psychic anxiety
cluster (Table 1). 5-HTTLPR variants were not associated with the remaining symptom clusters. We repeated
the ANOVA considering subgroups homogeneous for
diagnosis, age-of-onset (⬍25 years) and gender. The
association was stronger among bipolars (anxiety score:
s = 4.34 vs 1 = 3.42, F = 8.98, d.f. = 1,128, P = 0.003),
among early onset subjects (n = 31, anxiety score:
s = 4.65 vs 1 = 3.19, F = 11.66, d.f. = 1,60, P = 0.001)
and, as a trend, among females (data not shown). Severity of illness may be associated with symptom scores,
and it could constitute a stratification bias. So we
repeated the ANOVA including indicators of severity
5-HTTLPR and depressive symptomatology
A Serretti et al
Table 1 HAMD scores and 5-HTTLPR alleles and genotypes. Means and standard deviations of factor scores are reported.
For allele analysis subjects were counted twice. The 5-HTTLPR short variant was marginally associated with higher psychic
anxiety scores
5-HTTLPR
Core
Insomnia
Activity
Psychic
anxiety
Somatic
anxiety
Delusion
HAMD total
Mean s.d.
Mean s.d.
Mean s.d.
Mean s.d.
Mean s.d.
Mean s.d.
Mean s.d.
155
109
264
13.75 (3.31)
13.88 (3.02)
13.8 (3.19)
4.21 (1.58)
4.35 (1.58)
4.27 (1.58)
4.79 (1.37)
4.76 (1.38)
4.78 (1.37)
3.57 (2.31)
4.22 (2.39)
3.84 (2.34)
3.88 (1.39)
3.76 (1.41)
3.83 (1.40)
3.48 (2.27)
4.02 (2.12)
3.70 (2.22)
28.06 (6.68)
29.28 (6.79)
28.56 (6.74)
ANOVA
df = 1,262
F
P
0.11
n.s.
0.52
n.s.
0.03
n.s.
7.11
0.008
0.49
n.s.
3.74
n.s.
2.09
n.s.
Genotypes
11
12
22
All
48
59
25
132
ANOVA
df = 2,129
F
P
Alleles
1
2
All
No.
13.46
14.22
13.48
13.8
(3.39)
(3.18)
(2.83)
(3.20)
0.91
n.s.
4.08
4.41
4.28
4.27
(1.60)
(1.57)
(1.62)
(1.58)
4.73
4.90
4.60
4.78
0.55
n.s
as covariate, in turn. We considered the number of
depressive episodes and, only for bipolars, of manic
episodes, length of disease, presence/absence of
delusional features (DMS-IV), duration of index episode and HAMD total score. None of the covariates significantly influenced the results.
Our results indicate that the 5-HTTLPR polymorphism was not associated with total depressive symptomatology in our sample. We also observed a marginal
association between both the s allele and the s-containing genotypes and anxiety symptomatology. The
association was stronger when considering only
bipolar and early onset subjects. This is in accordance
with current views of a greater genetic loading in
those subjects.13
Abnormalities in the serotonin system have been
described in the pathogenesis of a number of behaviors
related to mood disorders.2 This led to the suggestion
that the influence of serotonin would be better analyzed at the level of symptoms rather than syndromes.
In fact Lesch et al6 detected a significant association
between the 5-HTTLPR genotype and the Neuroticism
factor in normals. Individuals with either one or two
copies of the s form had higher Neuroticism scores
than did individuals homozygous for the l variant.
However conflicting replications followed, with negative findings using both 5-HTTLPR and the 5HTT
VNTR,14 and with a positive association using the
5HTT VNTR.15 Mood disorders too were not unequivocally associated with both polymorphisms at 5HTTLPR,4,5,16,17 and a linkage study on bipolar families
using the PstI RFLP at the 5HTT locus did not support
the presence of a susceptibility locus for bipolar disorder near the serotinin transporter.18 Finally, the Tridimensional Personality Questionnaire temperament
of harm avoidance did not reveal associations of personality trait in normals with 5-HTTLPR.19
(1.35)
(1.41)
(1.35)
(1.37)
0.46
n.s.
3.19
4.20
4.24
3.84
(1.75)
(2.03)
(1.94)
(1.96)
4.39
n.s.
3.81
4.0
3.48
3.83
(1.45)
(1.30)
(1.50)
(1.40)
1.23
n.s.
3.38
3.66
4.44
3.70
(2.37)
(2.15)
(2.04)
(2.23)
1.93
n.s.
27.08
29.64
28.84
28.56
(6.46)
(6.84)
(6.85)
(6.75)
1.96
n.s.
Our study suggests an involvement of 5-HTTLPR
variants in anxiety symptomatology, among mood disorder subjects. Even though the main finding of this
paper is an absence of association with total depressive
symptomatology, together with our previous reports
excluding a 5-HTTLPR influence on symptoms of
major psychoses,8–10 we may hypothesize a broad effect
of 5-HTTLPR variants on anxiety symptomatology both
among patients and normals.6 Moreover both ls and ss
genotypes showed similar anxiety scores, in accordance with a putative dominant effect of the variant on
the serotonin transporter activity.6 This association
may explain previous conflicting results where anxiety
symptomatology was not considered, and a variable
rate of anxious subjects may have obscured or emphasized associations vs controls. Indeed, by selecting subjects with anxiety symptom scores one standard deviation above average or more, we observed that the rate
of s subjects rises from 0.41 to 0.54.
The power of our sample was enough to detect a
standardized difference (effect size) up to d = 0.35
(depending on the frequency of the 5-HTTLPR risk
allele, considering a power of 0.8 and alpha ⬍0.05 twotailed), that corresponds to a difference of 0.8 points
on the anxiety HAMD cluster score. Therefore our sample had sufficient power to detect the observed association. The variance explained by the s allele on the
anxiety factor is about 2%,20 which is in accordance
with current theories of polygenic inheritance in psychiatric disorders.21,22
Our sample is composed mainly by severe inpatients
as evidenced by a number of severity indicators like
total HAMD scores, number of episodes and the rate of
psychotic symptoms. This should indeed be considered a selection bias, probably due to the fact that
our center is a tertiary care setting. On the other hand
the selection of severe subjects should guarantee
281
5-HTTLPR and depressive symptomatology
A Serretti et al
282
against the possible inclusion of phenocopies.13 A limitation of the present study is the cross-sectional
approach, where we employed single time-point prevalence to rate depressive symptoms, which may not correspond to a lifetime perspective. However, we scored
subjects at index episode that correspond to a major
depressive episode requiring hospitalization, and follow-up studies evidenced that episodes are substantially stable over time both as for subtype and severity.23 Anyway, we cannot exclude future symptom
fluctuations. Another limitation might be related to
effects of drug treatments, but we specifically allowed
only low doses of benzodiazepines (flurazepam 15–
30 mg), with no antidepressant or neuroleptic treatments for at least 2 weeks. Moreover, the low doses
of benzodiazepines were administered to virtually all
subjects (95%) and only at bedtime. Ethnic origin is
frequently a cause of stratification bias but our sample
was composed of subjects with Italian antecedents for
at least two generations; though genetic heterogeneity
has been evidenced for some isolated populations,24
Italy is characterized by a substantial genetic homogeneity.25,26 Most studies, including the present, use multiple statistical testing. Therefore, significance levels
would be unlikely to survive correction in many cases.
Indeed, we corrected for the six HAMD factors and part
of our results survived this correction. However, for
analyses of candidate genes that have high a priori
probabilities of association, multiple correction could
not represent the best choice.27,28 Replication using
independent samples should be considered a more
suitable strategy.
Since the serotonin transporter is the major determinant of serotonin inactivation following release at synapses and it is the site of action of SSRI antidepressants, its activity should be better analyzed evaluating
response to SSRI antidepressants. In fact, while longterm adaptations may intervene among individuals
with the 5-HTTLPR short variant preventing any abnormal depressive symptomatology, during the rapid neurotransmitter changes while under antidepressant
treatment, s individuals may show an impaired
response to antidepressants. Our group is currently
testing this hypothesis. In conclusion, 5-HTTLPR was
marginally associated with psychic anxiety symptomatology in mood disorders; neither the remaining symptom clusters nor the total HAMD score were associated
with 5-HTTLPR variants.
Materials and methods
Sample
One hundred and thirty-two psychiatric inpatients
(age = 48.14 ± 13.26 years; age-of-onset = 34.74 ± 11.82
years; males 34.1%; education = 9.36 ± 3.95 years; duration of index epidode at admission = 16.7 ± 20.0
weeks) consecutively admitted to the Department of
Neuropsychiatry at the Institute H San Raffaele (DSNPHSR), were included in this study. Thirty-eight subjects are overlapping with a larger sample tested for a
possible interaction between the dopamine receptor D4
gene and 5-HTTLPR on psychotic symptomatology and
with a mood disorder sample scored by the OPCRIT
checklist.8,10 Patients were evaluated using the SADS29
and/or OPCRIT checklist.11 Lifetime diagnoses were
assigned by two independent psychiatrists on the basis
of interviews and medical records, according to DSMIV criteria.30 The Hamilton Depression Rating Scale
(HAMD-21)12 was administered at admission prior to
treatment by a trained psychiatrist and supervised by
a senior psychiatrist both blind to genetic data. Criteria
for inclusion in the study were: major depressive disorder, not including dysthymia (n = 67; age = 47.31 ±
13.09; onset = 35.66 ± 12.00; males 28.4%; No. depressive episodes = 4.53 ± 6.12; delusional subjects 38.8%)
and bipolar disorder, not including cyclothymia (n =
65; age = 49.0 ± 13.49; onset = 33.78 ± 11.65; males
40.0%; No. manic episodes = 2.23 ± 2.35; No. depressive episodes = 4.23 ± 2.87; delusional subjects 54.7%).
The presence of concomitant diagnoses of mental retardation or drug dependence, together with somatic or
neurological illnesses that impaired psychiatric evaluation (eg hypothyroidism mimicking a depressive state)
represented exclusion criteria. The only prior treatment permitted was low dose benzodiazepines (⬍5 mg
diazepam or equivalent); no prior (⬍2 weeks) antidepressant or neuroleptic treatment was allowed. Informed consent was obtained from all probands, who
were unrelated and of Italian descent with antecedents
from all parts of the country.
DNA analysis
DNA was extracted from leucocytes by NaCl precipatation.31 For PCR forward 5′-GGCGTTGCCGCT
CTGAATGC-3′ and reverse 5′-GAGGGACTGAGCTGGA
CAACCAC-3′ primers were employed. Thirty-five
cycles of 1 min at 95°C, 1 min at 61°C and 1 min at
72°C were performed. The assay mix contained in a
volume of 30 ␮l 50 ng genomic DNA, 2.5 mM dNTPs,
0.1 ␮g of sense and antisense primer, 10 mM Tris-HCl
(pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 5% DMSO and 1
U Taq Polymerase. PCR products were separated on
3% agarose gels supplemented with ethidium bromide
allowing differentiation of the long (528 bp) and the
short (484 bp) variant.
Statistical analysis
Differences were assessed by one-way Analysis of Variance (ANOVA) and Analysis of Covariance (ANCOVA)
when including covariates. Frequencies were compared by Chi-square test. Alpha values were considered significant when below 0.008 (0.05 / 6 HAMD
factors). HAMD items were pooled into the following
factors: Core (items 1, 2, 7, 8, 10, 13), Sleep (items 4,
5, 6), Activity (items 7, 8), Psychic anxiety (items 9,
10), Somatic anxiety (items 11, 12, 13), Delusion (items
2, 15, 20).32–37 The power of our sample for detecting
differences amongst 5-HTTLPR variants was calculated
considering an alpha value of 5% two-tailed. With
these parameters in our sample we had a high power
5-HTTLPR and depressive symptomatology
A Serretti et al
(0.80) to detect a medium–small effect size (0.35) that
corresponded to a difference of approximately 2.1
points between the two alleles on the total HAMD.20
19
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Correspondence: A Serretti, MD, Dept of Neuroscience, Istituto Scientifico H San Raffaele, University of Milan, School of Medicine, Via
Luigi Prinetti 29, 20127 Milano, Italy. E-mail: serretti.alessandro얀
mail.hsr.it
Received 27 July 1998; revised and accepted 9 September 1998
283