Dement Geriatr Cogn Disord 2016;41:334–347

DOI: 10.1159/000447324 © 2016 S. Karger AG, Basel

Accepted: June 1, 2016 1420–8008/16/0416–0334$39.50/0
Published online: July 15, 2016 www.karger.com/dem

Original Research Article

Association Study and Meta-Analysis

of Polymorphisms, Methylation Profiles,
and Peripheral mRNA Expression of the
Serotonin Transporter Gene in Patients
with Alzheimer’s Disease
Kiyohiro Yamazaki Yuta Yoshino Takaaki Mori Mitsuo Okita Taku Yoshida
Yoko Mori Yuki Ozaki Tomoko Sao Jun-ichi Iga Shu-ichi Ueno
Department of Neuropsychiatry, Molecules and Function, Ehime University Graduate School of
Medicine, Shitsukawa, Toon, Japan

Key Words
Alzheimer’s disease · Serotonin transporter · mRNA · Epigenetics · Apathy · Meta-analysis

Abstract
Background/Aim: The aim of this study was to elucidate the relationship between Alzheim-
er’s disease (AD) and the serotonin transporter gene (SLC6A4). Methods: AD subjects (n = 43)
and controls (n = 47) were recruited and evaluated. In leukocytes, we evaluated two polymor-
phisms in SLC6A4, the serotonin transporter length polymorphic region (5-HTT-LPR) and
rs25531, as well as methylation rates of the SLC6A4 promoter region and the SLC6A4 mRNA
expression level. We also performed a meta-analysis to examine the relationship between the
frequency of the L allele and the risk of AD. Results: The distributions of 5-HTT-LPR and
rs25531 polymorphisms in AD subjects were not different from those of controls. Although
the methylation rates in AD subjects were not significantly different from those of controls,
the expression level in AD subjects was significantly higher than in controls. Additionally, the
expression level in AD subjects was significantly correlated with apathy. Meta-analysis re-
vealed that the L/L genotype significantly reduced the risk of AD, but only in the Caucasian
population. Conclusion: Higher SLC6A4 mRNA expression in leukocytes in AD was associated
with apathy regardless of SLC6A4 genotypes and methylation rates of the promoter region.
The L/L genotype may reduce the risk of AD in the Caucasian population.
© 2016 S. Karger AG, Basel
Vanderbilt University - Eskind Biomedical Library
129.59.95.115 - 9/13/2016 3:32:57 AM

Jun-ichi Iga
Department of Neuropsychiatry, Molecules and Function
Ehime University Graduate School of Medicine
Shitsukawa, Toon, Ehime 791-0295 (Japan)
E-Mail igajunichi @ hotmail.com
Downloaded by:
 Dement Geriatr Cogn Disord 2016;41:334–347 335
DOI: 10.1159/000447324 © 2016 S. Karger AG, Basel
www.karger.com/dem
Yamazaki et al.: Polymorphisms, Methylation Profiles, and Peripheral mRNA Expression
of the Serotonin Transporter Gene in Patients with Alzheimer’s Disease

Introduction

Alzheimer’s disease (AD) is a chronic neurodegenerative disease and is characterized by

cognitive impairment and psychological symptoms including depression, apathy, delusion,
and other symptoms that are due to aggregation of amyloid plaques and neurofibrillary
tangles. Because of reduced levels of acetylcholine in the brain of AD patients, acetylcholines-
terase inhibitors (AChEIs) have been used as a first-line treatment for AD [1, 2]. However, the
progression of AD cannot be stopped even if treatment with AChEIs continues. Therefore, a
fundamental therapeutic agent based on the original causes of AD is needed.
Serotonin is a fundamental neurotransmitter that regulates cognitive functions, emotions,
interests, feelings, and other behaviors [3, 4]. The cell bodies of serotonergic neurons are
located in the raphe nuclei of the brainstem, and the nerve fibers are distributed throughout
the central nervous system [3, 5]. Therefore, many psychotropic drugs were developed to
modulate serotoninergic neurotransmission, particularly serotonin transporters encoded by
the serotonin transporter gene (SLC6A4; chromosomal location 17q12), which is a primary
target of antidepressants. SLC6A4 has frequently been associated with major depressive
disorder (MDD). A polymorphism upstream of the transcription start region in SLC6A4, called
the serotonin transporter length polymorphic region (5-HTT-LPR), is classified into two
common genotypes. The short (S) allele has 14 repeat sequences, and the long (L) allele has
16 repeats that consist of 20–24 nucleotides [6]. The S allele is associated with environmental
risk factors for MDD pathogenesis [7]. Nakamura et al. [8] identified a single nucleotide
variant (A to G) that divides the L allele into long A (LA) and long G (LG) alleles; these variants
were described as rs25531 by Kraft et al. [9]. Hu et al. [10] reported that the LA allele is asso-
ciated with high SLC6A4 mRNA transcription levels in vitro and that the LG allele is associated
with low mRNA transcription levels, similar to the S allele. Additionally, less common variants
of 5-HTT-LPR named extra-short alleles (XS; 11–13 repeat sequences) and extra-long alleles
(XL; 17–24 repeat sequences) have also been identified. The expression level of the XS alleles
is slightly lower than that of the S alleles, and expression of the XL alleles is not significantly
different from that of the L alleles [11, 12].
The transcriptional regulatory region of SLC6A4 contains a CpG island that affects its
mRNA expression level due to differences in methylation levels. Several studies have examined
the relationships between methylation rates of the SLC6A4 promotor region and SLC6A4
mRNA expression levels. Philibert et al. [13] reported that higher SLC6A4 promotor methyl-
ation is related to lower SLC6A4 mRNA expression levels in lymphoblast cell lines in Caucasian
samples. On the other hand, Wankerl et al. [14] reported that SLC6A4 mRNA expression levels
are unlikely to be affected by methylation profiles of the SLC6A4 promotor region.
Several studies showed that 5-HTT-LPR genotypes are associated with neuropsychiatric
symptoms in AD [15–20], whereas other studies did not replicate associations of 5-HTT-LPR
polymorphisms with neuropsychiatric symptoms [21–28]. Although previous studies consis-
tently demonstrated that SLC6A4 mRNA levels in leukocytes are significantly higher in
patients with MDD compared to healthy controls [29, 30], the mRNA expression level in
leukocytes from AD patients has not been examined yet. Thomas et al. [31] reported that
expression of serotonin transporters in the prefrontal cortex is lower in postmortem AD
brains from patients both with and without depression than in brains of healthy subjects.
Chen et al. [32] examined protein and mRNA levels of the serotonin transporter in post-
mortem prefrontal cortex and showed that levels are reduced significantly in AD compared
Vanderbilt University - Eskind Biomedical Library

with controls. These results in the brain are not consistent with our results in leukocytes. The
discrepancy may be derived from differences in tissues and ethnicities. Ethnic differences
129.59.95.115 - 9/13/2016 3:32:57 AM

were seen in allele frequencies between European and Japanese individuals. In cohorts of
European ancestry, the S allele was carried by 42–57% of individuals, whereas in Asian
Downloaded by:
 Dement Geriatr Cogn Disord 2016;41:334–347 336
DOI: 10.1159/000447324 © 2016 S. Karger AG, Basel
www.karger.com/dem
Yamazaki et al.: Polymorphisms, Methylation Profiles, and Peripheral mRNA Expression
of the Serotonin Transporter Gene in Patients with Alzheimer’s Disease

Table 1. Demographic data and

Control AD p
clinical characteristics of control
(n = 47) (n = 43) value
and AD subjects
Male (%) 16 (34.0) 16 (37.2) 0.240
Age, years 79.7 ± 4.41 79.8 ± 3.84 0.876
Duration of illness, months 67.16 ± 59.17
MMSE score 18.04 ± 5.30
CDR score 1.34 ± 0.75
NPI (12 items) score 13.47 ± 16.36

Sex: χ2 test; age: Student’s t test. Data are given as mean ± SD unless
indicated otherwise.

cohorts, the S allele was carried by 75–89% of AD patients [33], and 82% of healthy Japanese
subjects carried the S allele [34]. Ouchi et al. [35] reported that striatal 11C-3-amino-4-benzo-
nitrile (11C-DASB), a specific serotonin transporter marker binding, is significantly lower in
AD patients, irrespective of depression, than in healthy controls, whereas 11C-DASB binding
in other dense projection areas is significantly decreased in the group with depression,
suggesting that a higher loss of serotonergic function is related to more severe psychiatric
symptoms in AD. Thus, AD pathogenesis can be influenced by SLC6A4. However, the relation-
ships among SLC6A4 polymorphisms, methylation rates, and the mRNA expression level in
leukocytes in AD have not been examined. Thus, the aim of our study was to investigate the
relationships among the genetic, epigenetic, and expression changes of SLC6A4 in peripheral
leukocytes and the psychological symptoms in Japanese AD subjects.

Methods

Subjects
Demographic data for each group of participants are shown in table 1. We enrolled 43 AD subjects [16
males and 27 females, mean age ± standard deviation (SD) = 79.8 ± 3.84 years] who visited Ehime University
Hospital or Zaidan Niihama Hospital, Ehime, Japan from December 2013 to May 2015. AD subjects were diag-
nosed and classified as having probable AD dementia according to the criteria of the National Institute on Aging
and the Alzheimer’s Association [36]. AD subjects did not take any antidepressants including selective serotonin
reuptake inhibitors, selective norepinephrine reuptake inhibitors, noradrenergic and specific serotonergic anti-
depressants, tricyclic antidepressants, or tetracyclic antidepressants at the time of blood sampling. AD subjects
were evaluated with the Mini Mental State Examination (MMSE) to assess their cognitive function [37], with the
Clinical Dementia Rating (CDR) measured by family caregivers [38], and with the Neuropsychiatric Inventory
(NPI) to assess their psychological symptoms [39]. Control participants were 47 elderly individuals (16 males
and 31 females, mean age ± SD = 79.7 ± 4.41 years) without cognitive impairment, psychiatric signs, or a past
history of mental disorders as determined by at least two certified psychiatrists based on clinical interviews. All
participants were unrelated, of Japanese origin, and provided written informed consent forms that were
approved by the institutional ethics committees of Ehime University Hospital and Zaidan Niihama Hospital.

Blood Sample Collection, Extraction of Genomic DNA, and Synthesis of Complementary DNA
Genomic DNA (gDNA) was obtained from whole peripheral blood samples collected in potassium EDTA
tubes and extracted using the QIAamp DNA Blood Mini Kit (Qiagen, Tokyo, Japan) according to the standard
Vanderbilt University - Eskind Biomedical Library

protocol. Total RNA was isolated from whole peripheral blood samples using PaxGene Blood RNA Systems
tubes (BD, Tokyo, Japan) according to the standard protocol. The RNA concentration and purity were
129.59.95.115 - 9/13/2016 3:32:57 AM

measured with a NanoDrop-1000 (Thermo Fisher Scientific, Yokohama, Japan), and the 260/280 ratio was
between 1.8 and 2.0. RNA (1.0 μg) in a 40-μl total reaction volume per sample was used to synthesize cDNA
with the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, Calif., USA).
Downloaded by:
 Dement Geriatr Cogn Disord 2016;41:334–347 337
DOI: 10.1159/000447324 © 2016 S. Karger AG, Basel
www.karger.com/dem
Yamazaki et al.: Polymorphisms, Methylation Profiles, and Peripheral mRNA Expression
of the Serotonin Transporter Gene in Patients with Alzheimer’s Disease

Fig. 1. The position of the 9 CpG sites in SLC6A4. The F and R primers amplified a region that included the 9
CpG sites. The S is the sequencing primer. Significant Spearman’s correlation coefficients (ρ) are shown be-
low for each CpG site in AD subjects.

Genotyping Analysis
For genotyping, PCR was performed with 12.5 ng gDNA, 0.2 μM each primer, 0.25 U KOD FX Neo (Toyobo
Co., Ltd.), 2× buffer for KOD FX Neo, and 2 mM dNTPs. Primer sequences were: 5′-GGCGTTGCCGCTCT-
GAATGC-3′ (forward primer) and 5′-GAGGGACTGAGCTGGACAACCAC-3′ (reverse primer) [6, 40]. Cycling
conditions were as follows: denaturation at 94 ° C for 2 min, followed by 30 cycles of 94 ° C for 10 s, 71 ° C for
30 s, and 72 ° C for 30 s. PCR fragments were digested with MspI restriction enzyme (New England Biolabs
Inc.), and the 5-HTT-LPR genotype was determined according to the size of the bands following agarose gel
electrophoresis. Product sizes following digestion were S allele = 297 bp, LA allele = 340 bp, LG allele = 166 +
174 bp, and common products of all polymorphisms = 62 and 127 bp [40–42]. The size of the XS alleles is
smaller than the S alleles, and the size of the XL alleles is larger than the L alleles.

Bisulfite Conversion and Pyrosequencing

The methylation status was analyzed in the region of the SLC6A4 promoter shown in figure 1. The
SLC6A4 promoter region includes 81 CpG sites in a 799-bp region [13]. According to a previous study showing
that one CpG-rich region in the promotor region affects SLC6A4 mRNA expression levels, we analyzed the
same region that includes 9 CpG sites in the transcription start site [13, 43] (fig. 1). The gDNAs extracted from
leukocytes were treated with bisulfate using the EpiTect Plus DNA Bisulfite Kit (Qiagen, Valencia, Calif., USA)
Vanderbilt University - Eskind Biomedical Library

and then amplified with PCR using the forward primer 5′-TTTTAGGAAGAAAGAGAGAGTAGTT-3′ and the
reverse primer 5′-[Biotin]-ATCCTAACTTTCCTACTCTTTAACT-3′. PCR was performed in a 40.0-μl final
129.59.95.115 - 9/13/2016 3:32:57 AM

volume per sample and included 2.5 μl gDNAs, 0.2 μM each primer, AmpliTaq gold (Applied Biosystems), 10×
PCR buffer with 15 mM MgCl2, and 2 mM dNTPs. Cycling conditions were as follows: denaturation at 94 ° C for
10 min, followed by 45 cycles of 94 ° C for 30 s, 54 ° C for 30 s, and 72 ° C for 1 min, with a final extension of 10
Downloaded by:
 Dement Geriatr Cogn Disord 2016;41:334–347 338
DOI: 10.1159/000447324 © 2016 S. Karger AG, Basel
www.karger.com/dem
Yamazaki et al.: Polymorphisms, Methylation Profiles, and Peripheral mRNA Expression
of the Serotonin Transporter Gene in Patients with Alzheimer’s Disease

min at 72 ° C. The PCR products were sequenced with PyroMark Q24 Advanced (Qiagen, Hilden, Germany)
using the sequencing primer 5′-AGTTTTAGGGATGGG-3′. Methylation rates at each CpG were quantified in
duplicate using PyroMark Q24 Advanced Software (Qiagen, Tokyo, Japan).

PCR Array Procedure

For mRNA expression analysis, real-time quantitative reverse transcription-PCR was performed using
the StepOnePlus Real-Time PCR System (Applied Biosystems). Specific TaqMan probes were Hs00984356_
m1 for SLC6A4 and Hs99999905_m1 for GAPDH. We used GAPDH as a housekeeping gene because previous
studies including ours consistently identified GAPDH as a suitable housekeeping gene for leukocyte gene
expression analysis using the Paxgene blood RNA system [44–47]. The final volume of the reactions was 10
μl and included the TaqMan Universal Master Mix (Applied Biosystems). The expression levels were examined
in triplicate. The ΔΔCt method was used to determine relative expression levels using StepOne software
(Applied Biosystems) [48]. To correct for observational errors, PCR was performed in all plates with gDNA
from the same control subjects in this study.

Meta-Analysis Methods
Data were collected from PubMed. The key words ‘serotonin transporter’ and ‘Alzheimer disease’ were
used to search for studies. Published studies to examine the association between the serotonin transporter
and AD were carefully selected by two independent investigators (J.I. and K.Y.). Case-control studies of AD
cases and healthy controls were included for further selection. The meta-analysis examined the relationship
between the frequency of the L allele and the risk of AD. Odds ratios (ORs) and their 95% confidence intervals
(95% CIs) were estimated for L/L versus S carriers (L/S and S/S). To combine individual study results, we
conducted the meta-analysis using EZR [49].

Statistical Analysis
Statistical analysis was performed using SPSS Statistics version 22.0 (IBM Corp., Tokyo, Japan). For
analysis of the effects of SLC6A4 alleles on each parameter, these alleles were divided into three groups
defined by the presence of the L allele or the LG allele in a ‘dose-dependent’ manner: S/S (dose = 0), L/S
(dose = 1), L/L (dose = 2), or S (LG)/S (LG) (dose = 0), LA/S (LG) (dose = 1), LA/LA (dose = 2) [50, 51]. SLC6A4
mRNA expression levels, methylation rates in each CpG site, and the participant’s age were compared
between AD and control subjects with the Student t test or Mann-Whitney U test after the Shapiro-Wilk
test. Sex differences and distributions of SLC6A4 alleles were compared with the χ2 test. Difference in
mRNA expression levels among SLC6A4 alleles were compared with the Kruskal-Wallis test. Correlations
among age, duration of illness, MMSE total scores, NPI total scores, CDR scores, mRNA expression levels,
and methylation rates as well as between SLC6A4 alleles and methylation rates were analyzed with the
Spearman’s correlation test. Relationships between mRNA expression levels and each psychological
symptom in NPI (presence of the symptom = 1, absence = 0) were analyzed with a multiple linear regression
analysis stepwise method. Additionally, the Mann-Whitney U test after the Shapiro-Wilk test was
conducted to compare the mRNA expression level and the apathy score (frequency × severity) in the NPI
subscale. Each psychological symptom in NPI and the distributions of SLC6A4 alleles were compared with
the χ2 test. Statistical significance was defined at the 95% level (p = 0.05) except for the 95% level (p =
0.005) after the Bonferroni correction for analysis of each methylation rate and the 95% level (p = 0.004)
after the Bonferroni correction for analysis of each psychological symptom in the NPI. Meta-analysis and
power calculations were performed using EZR (Saitama Medical Center, Jichi Medical University, Saitama,
Japan) [49].

Results

Participant Characteristics and Genotyping

Vanderbilt University - Eskind Biomedical Library

The demographic data of the participants are shown in table 1. AD and control subjects
did not differ in gender or age. Clinical characteristics and the results of psychological tests
129.59.95.115 - 9/13/2016 3:32:57 AM

in AD subjects are also shown in table 1. Only one control subject had the extra-long A allele
and was included in the group with the LA allele according to previous studies [10, 11]. The
Downloaded by:
 Dement Geriatr Cogn Disord 2016;41:334–347 339
DOI: 10.1159/000447324 © 2016 S. Karger AG, Basel
www.karger.com/dem
Yamazaki et al.: Polymorphisms, Methylation Profiles, and Peripheral mRNA Expression
of the Serotonin Transporter Gene in Patients with Alzheimer’s Disease

Table 2. Distributions of SLC6A4 alleles

Alleles S or L type, n (%) l or h type, n (%)

S/S L/S L/L p value l/l h/l h/h p value

Total (n = 90) 56 (62.2) 29 (32.2) 5 (5.6) 64 (71.1) 25 (27.8) 1 (1.1)

Control (n = 47) 32 (68.1) 12 (25.5) 3 (6.4) 35 (74.5) 11 (23.4) 1 (2.1)
AD (n = 43) 24 (55.8) 17 (39.5) 2 (4.7) 0.387 29 (67.4) 14 (32.6) 0 (0) 0.480

The short allele of 5-HTT-LPR is denoted ‘S’ type, and the long allele is denoted ‘L’ type. The L allele with the G variant for
rs25531 or the S allele is shown as ‘l’ type, and the L allele with the A variant is shown as ‘h’ type. Differences in the ratio for
each genotype between control and AD subjects were not significant according to the χ2 test.

18
Control
16 AD

14
Methylation rates of SLC6A4

12

10

0
CpG1 CpG2 CpG3 CpG4 CpG5 CpG6 CpG7 CpG8 CpG9 CpG mean

Fig. 2. Methylation rates at each CpG site in AD and control subjects. The data are the mean + SE. No signifi-
cant differences were found.

distributions of 5-HTT-LPR and rs25531 are shown in table 2. The ratio of each allele in the
two classified groups in AD was not significantly different from those of control subjects
(5-HTT-LPR genotypes, p = 0.387; alleles with rs25531 polymorphisms, p = 0.480).

Methylation Status
The sequence and the position of the 9 CpG sites in the SLC6A4 promotor region are
depicted in figure 1. Methylation rates at each CpG site are shown in figure 2. The methylation
rates at almost all CpG sites were correlated with each other in AD and control subjects (fig. 1).
Methylation rates at individual CpG sites were not correlated with gender, age at blood
Vanderbilt University - Eskind Biomedical Library

sampling, the 5-HTT-LPR genotypes, or alleles with the rs25531 polymorphism in either AD
or control subjects. Additionally, methylation rates at individual CpG sites were not corre-
129.59.95.115 - 9/13/2016 3:32:57 AM

lated with the duration of illness in AD subjects. We found no significant differences between
the methylation rate at each CpG site in AD subjects and those in control subjects (fig. 2).
Downloaded by:
 Dement Geriatr Cogn Disord 2016;41:334–347 340
DOI: 10.1159/000447324 © 2016 S. Karger AG, Basel
www.karger.com/dem
Yamazaki et al.: Polymorphisms, Methylation Profiles, and Peripheral mRNA Expression
of the Serotonin Transporter Gene in Patients with Alzheimer’s Disease

10 *

SLC6A4 expression level

8

Fig. 3. SLC6A4 mRNA expression 4

levels in AD and control subjects.
The mean expression level in AD 2
subjects (3.20 ± 1.86) was signifi-
cantly higher than that in control 0
Control (n = 47) AD (n = 43)
subjects (2.58 ± 1.70) (Mann-
Whitney U test, * p = 0.046).

Table 3. Correlations between

mRNA MMSE CDR NPI
the SLC6A4 mRNA expression
expression level (12 items)
level and total scores of
psychological tests in AD subjects
r −0.065 0.195 0.377
p value 0.677 0.211 0.013*

The NPI total score was correlated with the SLC6A4 mRNA expression
level. r = Spearman’s correlation coefficient. * p < 0.05.

mRNA Expression Level

SLC6A4 mRNA expression levels were not associated with gender (AD subjects, p = 0.191;
control subjects, p = 0.12), age at blood sampling (AD, p = 0.969; control, p = 0.772), 5-HTT-LPR
genotype (AD, p = 0.097; control, p = 0.714), or alleles with the rs25531 polymorphism (online
suppl. fig. 1; for all online suppl. material, see www.karger.com/doi/10.1159/000447324;
AD, p = 0.407, online suppl. fig. 2; control, p = 0.272) in either AD or control subjects or with
duration of illness in AD subjects (p = 0.761). The SLC6A4 mRNA expression level in AD
subjects was significantly higher than that in controls (fig. 3; p = 0.046). The SLC6A4 mRNA
expression level in AD subjects treated with AChEIs (n = 12) was not significantly different
from those not treated with AChEIs (n = 31) (p = 0.357).

Relationships between the SLC6A4 mRNA Expression Level and Scores in the MMSE, CDR,
and NPI and between the Methylation Rates and SLC6A4 Alleles and Psychological Scores
Correlations between the SLC6A4 mRNA expression level and each psychological test in
AD subjects are shown in table 3. The SLC6A4 mRNA expression level in AD subjects was
correlated with the NPI total score (table 3; p = 0.013) and was significantly associated with
apathy in the NPI following the multiple linear regression analysis stepwise method (p =
0.03). Additionally, the SLC6A4 mRNA expression level in AD subjects with apathy (n = 29)
was significantly higher than in those without apathy (n = 14, p = 0.02; fig. 4). We found no
correlation between the SLC6A4 mRNA expression level and MMSE total scores or CDR scores
(table 3). The methylation rates in AD subjects were not correlated with the 5-HTT-LPR
Vanderbilt University - Eskind Biomedical Library

genotype, SLC6A4 allele with the rs25531 polymorphism, the SLC6A4 mRNA expression level,
MMSE total scores, CDR scores, or NPI total scores (table 4). In addition, the 5-HTT-LPR
129.59.95.115 - 9/13/2016 3:32:57 AM

genotype and SLC6A4 allele with the rs25531 polymorphism were not associated with any of
the psychological symptoms in NPI.
Downloaded by:
 Dement Geriatr Cogn Disord 2016;41:334–347 341
DOI: 10.1159/000447324 © 2016 S. Karger AG, Basel
www.karger.com/dem
Yamazaki et al.: Polymorphisms, Methylation Profiles, and Peripheral mRNA Expression
of the Serotonin Transporter Gene in Patients with Alzheimer’s Disease

10 *

SLC6A4 expression level

8

6
Fig. 4. SLC6A4 mRNA expression
level with or without apathy. The 4
data are the mean + SE. The
SLC6A4 mRNA expression level in 2
AD subjects with apathy (n = 29)
was significantly higher than in 0
No apathy (n = 14) Apathy (n = 29)
those without apathy (n = 14;
Mann-Whitney U test, * p = 0.02).

Table 4. Correlations between the methylation rate at each CpG site in AD subjects and SLC6A4 alleles, the
SLC6A4 mRNA expression level, MMSE total scores, CDR scores, and NPI total scores

AD methylation Baseline score

site
alleles alleles expression MMSE CDR NPI
(S or L type) (l or h type) level

CpG1 −0.002 0.072 −0.056 −0.282 0.164 0.102

CpG2 0.182 0.008 −0.20 0.309 −0.165 −0.21
CpG3 0.072 0.042 −0.320 0.129 0.017 −0.217
CpG4 0.154 0.028 −0.199 0.136 0.081 0.002
CpG5 0.02 −0.036 −0.02 0.028 0.114 0.042
CpG6 0.034 −0.038 −0.14 0.067 0.078 0.125
CpG7 0.248 0.172 −0.134 −0.088 0.156 0.074
CpG8 0.012 −0.068 −0.108 0.117 −0.057 0.122
CpG9 −0.1 −0.12 −0.126 −0.197 0.096 0.12
CpG mean 0.096 −0.028 −0.178 −0.051 0.056 0.095

Spearman’s correlation coefficients are shown. We found no significant correlations at any CpG site (p ≤
0.005 after Bonferroni correction was considered significant).

Results of Meta-Analysis
We identified 27 potentially relevant papers, but only 15 met the inclusion criteria. The
12 excluded papers included 6 studies that did not examine healthy controls, and 6 reviews
and brief reports. Five case-control datasets were included in the meta-analysis of the Asian
population, which included 524 cases and 699 controls [24, 26, 52, 53]. Ten case-control
datasets were included in the meta-analysis of the Caucasian population, which included
1,436 cases and 1,342 controls. We tested the association between 5-HTT-LPR and AD by
estimating the ORs for L/L versus S carriers (L/S and S/S) [17, 21, 22, 25, 54–59]. Interest-
ingly, although we identified no significant difference between the L/L genotype and S carriers
in the Asian population (fig. 5; OR L/L vs. S carrier = 1.22; 95% CI, 0.71–2.11), the L/L genotype
Vanderbilt University - Eskind Biomedical Library

was associated with a significantly reduced risk of development of AD in the Caucasian popu-
lation (fig. 6; OR L/L vs. S carrier = 0.78; 95% CI, 0.66–0.92). For meta-analysis, we identified
129.59.95.115 - 9/13/2016 3:32:57 AM

14 studies that examined the association between 5-HTT-LPR and any behavior and psycho-
logical symptoms of dementia (online suppl. table 1). Unfortunately, we could not conduct a
Downloaded by:
 Dement Geriatr Cogn Disord 2016;41:334–347 342
DOI: 10.1159/000447324 © 2016 S. Karger AG, Basel
www.karger.com/dem
Yamazaki et al.: Polymorphisms, Methylation Profiles, and Peripheral mRNA Expression
of the Serotonin Transporter Gene in Patients with Alzheimer’s Disease

First author [Ref.] Experimental Control OR [95% CI] W W

(fixed), (random),
events total events total
% %

Kunugi [52] 4 89 15 326 1.09 [0.35, 3.38] 24.2 24.3

Tsai [53] 16 136 6 83 1.71 [0.64, 4.56] 28.3 32.3
Ha [24] 3 65 3 43 0.65 [0.12, 3.36] 14.9 11.4
Ueki [26] 7 200 5 200 1.41 [0.44, 4.53] 20.8 22.9
This study 2 43 3 47 0.72 [0.11, 4.50] 11.8 9.2

Fixed effect model 524 699 1.22 [0.71, 2.11] 100.0 –

Random effects model 1.21 [0.60, 2.12] – 100.0
Heterogeneity: I2 = 0%, τ2 = 0, p = 0.8348
0.75 1.5
OR

Fig. 5. Forest plots for meta-analysis in the Asian population. Pooled ORs and 95% CIs were examined for
L/L versus S carriers (L/S and S/S).

First author [Ref.] Experimental Control OR [95% CI] W W

(fixed), (random),
events total events total
% %

Fehér [54] 79 252 72 234 1.03 [0.70, 1.51] 15.7 12.8

Lorenzi [55] 49 164 26 54 0.46 [0.24, 0.86] 8.4 8.7
Seripa [56] 39 105 33 114 1.45 [0.82, 2.55] 6.1 9.6
Micheli [25] 66 208 39 116 0.92 [0.57, 1.49] 10.5 11.0
Borroni [17] 16 82 52 152 0.47 [0.25, 0.88] 9.0 8.5
Nishimura [57] 34 128 48 126 0.59 [0.35, 1.00] 10.9 10.2
Rocchi [22] 50 135 31 90 1.12 [0.64, 1.96] 7.2 9.8
Zill [58] 28 85 40 118 0.96 [0.53, 1.73] 6.9 9.2
Oliveira [59] 18 81 36 81 0.36 [0.18, 0.71] 8.6 8.0
Li [21] 47 196 83 257 0.66 [0.43, 1.01] 16.7 12.2

Fixed effect model 1,436 1,342 0.78 [0.66, 0.92] 100.0 –

Random effects model 0.75 [0.58, 0.98] – 100.0
Heterogeneity: I2 = 57.2%, τ2 = 0.0966, p = 0.0126
0.75 1 1.5
OR

Fig. 6. Forest plots for meta-analysis in the Caucasian population. Pooled ORs and 95% CIs were examined
for L/L versus S carriers (L/S and S/S).

meta-analysis between 5-HTT-LPR and depression or apathy in AD, because ethnicity and
assessments were diverse and most studies did not provide the data on depression or apathy
in detail.

Discussion
Vanderbilt University - Eskind Biomedical Library

The frequencies of the 5-HTT-LPR and rs25531 polymorphisms in AD subjects were not
129.59.95.115 - 9/13/2016 3:32:57 AM

different from those of control subjects in our study. The frequency of the 5-HTT-LPR genotype
[60, 61] and the frequency of the rs25531 polymorphism [8] were consistent with previous
Downloaded by:
 Dement Geriatr Cogn Disord 2016;41:334–347 343
DOI: 10.1159/000447324 © 2016 S. Karger AG, Basel
www.karger.com/dem
Yamazaki et al.: Polymorphisms, Methylation Profiles, and Peripheral mRNA Expression
of the Serotonin Transporter Gene in Patients with Alzheimer’s Disease

studies in Japanese or Asian subjects. Hu et al. [10] reported that the LG alleles are associated
with a higher SLC6A4 expression in lymphoblast cell lines. However, we found no genotype
effects on SLC6A4 mRNA expression in peripheral leukocytes. Consistent with this obser-
vation, Martin et al. [62] did not find an effect of the LG variant on SLC6A4 expression. Therefore,
differences in frequencies of the variants, sample size, and experimental setting (in vitro vs.
in vivo) may have an influence on the results. Moreover, a previous study used positron
emission tomography in conjunction with 11C-DASB to examine the availability of the sero-
tonin transporter in 7 brain regions in 63 healthy European Caucasian volunteers who were
genotyped for 5-HTT-LPR and rs25531 and revealed that these polymorphisms did not affect
the availability of the serotonin transporter in adult human brain [63]. Therefore, we suggest
that the role of genetic variation at this locus in regulating the mRNA level may be complex in
vivo, and that the effect size of the LG allele on mRNA expression may be smaller than origi-
nally suggested. We performed the first comprehensive meta-analysis to examine the rela-
tionship between the 5-HTT-LPR genotype and the development of AD. Our results demon-
strated a significant association between 5-HTT-LPR and AD, but only in the Caucasian popu-
lation. Our findings indicate that population differences should be considered when examining
this polymorphism and that the S allele plays an important role not only in depression [7, 34,
64] but also in AD, particularly in the Caucasian population.
Neuropsychiatric symptoms in AD may be associated with 5-HTT-LPR. Borroni et al. [17]
reported that the 5-HTT-LPR S allele is associated with psychosis. Pritchard et al. [18] and
Proitsi et al. [20] reported that the 5-HTT-LPR S allele is associated with irritability. Quaranta
et al. [19] demonstrated that the risk of psychosis is increased in individuals with the
5-HTT-LPR L allele, and Sukonick et al. [15] and Sweet et al. [16] reported that the L allele is
significantly associated with aggression in AD. Conversely, other studies did not find signif-
icant associations between 5-HTT-LPR polymorphisms and neuropsychiatric symptoms in
AD [21–28] (online suppl. table 1). However, the influence of all psychiatric symptoms was
not assessed and cannot be excluded, because these studies used various methods for the
assessment of neuropsychiatric symptoms. Our meta-analysis indicated the presence of
ethnic differences in these genotypes: 21.6–28.3% of Caucasians and 55.6–60.0% of Asians
have the S/S genotype, and 29–43% of Caucasians and 1–13% of East Asians have the L/L
genotype [60, 65–67]. Therefore, we should consider ethnic differences and be aware that the
results of our study may not be adapted to Caucasians. Consistent with our results, Ueki et al.
[26] and Ha et al. [24] found that psychotic symptoms are not associated with 5-HTT-LPR
genotypes. Although the consistent results in Asian studies are interesting, the discrepancies
may be due to the small sample sizes in these studies, including ours.
Methylation rates at CpG1 and CpG9 in our study were relatively higher than rates of
other CpGs, consistent with previous studies examining the same CpG sites [68, 69]. However,
the mean methylation rates of the 9 CpG sites in our study were higher than those in preceding
studies [68–72]. Domschke et al. [69] reported that methylation rates of the same CpG sites
increase with increasing age. Thus, an effect of aging may be present on methylation rates in
our study in which the mean age of participants was more than 25 years older than the mean
age of participants in the previous study. Okada et al. [73] found no significant difference
between unmedicated patients with MDD and healthy control subjects for any methylation
rate of SLC6A4 promoter CpG sites. Meanwhile, Philibert et al. [74] reported a trend for an
increased overall methylation rate in patients with a lifetime history of major depression.
Unfortunately, ours is the first study showing no association between AD and the methylation
Vanderbilt University - Eskind Biomedical Library

rates of the SLC6A4 promoter. Therefore, further studies are warranted to confirm the finding.
Our study revealed that the SLC6A4 mRNA expression level in AD subjects was signifi-
129.59.95.115 - 9/13/2016 3:32:57 AM

cantly higher than that in control subjects. This is the first study showing an elevated SLC6A4
mRNA expression level in leukocytes from AD patients. Although the result of our study was
Downloaded by:
 Dement Geriatr Cogn Disord 2016;41:334–347 344
DOI: 10.1159/000447324 © 2016 S. Karger AG, Basel
www.karger.com/dem
Yamazaki et al.: Polymorphisms, Methylation Profiles, and Peripheral mRNA Expression
of the Serotonin Transporter Gene in Patients with Alzheimer’s Disease

not consistent with previous studies performed by Thomas et al. [31] or Chen et al. [32] who
examined postmortem AD brains, we should consider differences in genotype and allele
frequencies, psychological symptoms, and tissues. Therefore, our results must be confirmed
in other races including Caucasians. In several studies, patients with MDD showed a higher
SLC6A4 mRNA expression level in leukocytes compared to healthy subjects [12, 29, 75].
Philibert et al. [74] reported that MDD subjects with a lifetime history of alcohol dependence
had higher SLC6A4 mRNA expression levels. Therefore, SLC6A4 mRNA expression may be
associated with various psychiatric symptoms accompanied by AD. Although an association
between antidepressant treatment and the SLC6A4 mRNA expression level was reported
earlier [12, 29], we found no association between treatment with AChEIs and SLC6A4 mRNA
expression levels in our study. This discrepancy may be due to differences in pharmacody-
namics (serotonin reuptake inhibition at the serotonin transporter vs. acetylcholine esterase
inhibition in the synaptic cleft). Interestingly, the SLC6A4 mRNA expression level in AD was
significantly correlated with NPI scores, especially apathy, among 12 subscores. In a post-
mortem brain study, Chen et al. [32] reported that the SLC6A4 mRNA expression level was
significantly decreased in the prefrontal cortex of AD subjects. Using 11C-Pittsburgh compound
B positron emission tomography, Mori et al. [76] reported that the bilateral prefrontal cortex
in patients with AD with apathy showed greater Aβ deposition compared to patients without
apathy. The higher SLC6A4 mRNA expression level in leukocytes of AD patients with apathy
may be associated with the lower SLC6A4 mRNA expression level and greater Aβ deposition
in the prefrontal cortex of AD.
Our study has several limitations. The first limitation is the small sample size. We
performed power calculations, and the sample size was not sufficient to detect differences in
the distribution of 5-HTT-LPR genotypes (required sample size estimated to be n = 1,099) or
alleles with the rs25531 polymorphism (n = 5,103). Therefore, we cannot conclude that
5-HTT-LPR is not associated with the development or symptoms of AD. A second limitation
is that potential influences of medication, food, and the patient’s physical condition on mRNA
expression levels and methylation rates were not examined.
In summary, our results suggest that higher SLC6A4 mRNA expression levels in leuko-
cytes of AD may be associated with apathy regardless of the SLC6A4 genotype and the meth-
ylation rates of the SLC6A4 promoter region. 5-HTT-LPR may play an important role in AD in
the Caucasian population. Further studies are warranted to confirm these findings.

Acknowledgements

We thank Zaidan Niihama Hospital for collecting blood samples and Ms. Chiemi Onishi for her technical
assistance. This work was partly supported by JSPS KAKENHI Grant Numbers 25861015 and 15K09808.

Disclosure Statement

The authors declare no conflicts of interest.

References
Vanderbilt University - Eskind Biomedical Library

 1 Jorm AF: Effects of cholinergic enhancement therapies on memory function in Alzheimer’s disease: a meta-
analysis of the literature. Aust NZ J Psychiatry 1986;20:237–240.
129.59.95.115 - 9/13/2016 3:32:57 AM

 2 Wilkinson DG, Francis PT, Schwam E, Payne-Parrish J: Cholinesterase inhibitors used in the treatment of
Alzheimer’s disease: the relationship between pharmacological effects and clinical efficacy. Drugs Aging 2004;
21:453–478.
Downloaded by:
 Dement Geriatr Cogn Disord 2016;41:334–347 345
DOI: 10.1159/000447324 © 2016 S. Karger AG, Basel
www.karger.com/dem
Yamazaki et al.: Polymorphisms, Methylation Profiles, and Peripheral mRNA Expression
of the Serotonin Transporter Gene in Patients with Alzheimer’s Disease

 3 Frazer A, Hensler JG: Molecular, cellular and medical aspects; in Siegel JS, Agranoff BW, Albers RW, Molinoff
PB (eds): Basic Neurochemistry, ed 5. Philadelphia, Lippincott-Raven, 1994, pp 283–308.
 4 Hornung JP: The human raphe nuclei and the serotonergic system. J Chem Neuroanat 2003;26:331–343.
 5 Fujita M, Shimada S, Maeno H, Nishimura T, Tohyama M: Cellular localization of serotonin transporter mRNA
in the rat brain. Neurosci Lett 1993;162:59–62.
 6 Heils A, Teufel A, Petri S, Stöber G, Riederer P, Bengel D, Lesch KP: Allelic variation of human serotonin trans-
porter gene expression. J Neurochem 1996;66:2621–2624.
 7 Karg K, Burmeister M, Shedden K, Sen S: The serotonin transporter promoter variant (5-HTTLPR), stress, and
depression meta-analysis revisited: evidence of genetic moderation. Arch Gen Psychiatry 2011;68:444–454.
 8 Nakamura M, Ueno S, Sano A, Tanabe H: The human serotonin transporter gene linked polymorphism
(5-HTTLPR) shows ten novel allelic variants. Mol Psychiatry 2000;5:32–38.
 9 Kraft JB, Slager SL, McGrath PJ, Hamilton SP: Sequence analysis of the serotonin transporter and associations
with antidepressant response. Biol Psychiatry 2005;58:374–381.
10 Hu XZ, Lipsky RH, Zhu G, Akhtar LA, Taubman J, Greenberg BD, Xu K, Arnold PD, Richter MA, Kennedy JL,
Murphy DL, Goldman D: Serotonin transporter promoter gain-of-function genotypes are linked to obsessive-
compulsive disorder. Am J Hum Genet 2006;78:815–826.
11 Ehli EA, Hu Y, Lengyel-Nelson T, Hudziak JJ, Davies GE: Identification and functional characterization of three
novel alleles for the serotonin transporter-linked polymorphic region. Mol Psychiatry 2012;17:185–192.
12 Seripa D, Iurescia S, Rinaldi M, Pilotto A, Paroni G, Bazzano S, Antonelli A, Gravina C, Urbano M, Padovani A,
Pilotto A: The serotonin transporter gene-linked polymorphic region: identification and functional character-
ization of novel alleles. Eur Geriatr Med 2013;4:S100.
13 Philibert R, Madan A, Andersen A, Cadoret R, Packer H, Sandhu H: Serotonin transporter mRNA levels are
associated with the methylation of an upstream CpG island. Am J Med Genet B Neuropsychiatr Genet 2007;
144B:101–105.
14 Wankerl M, Miller R, Kirschbaum C, Hennig J, Stalder T, Alexander N: Effects of genetic and early environ-
mental risk factors for depression on serotonin transporter expression and methylation profiles. Transl
Psychiatry 2014;4:e402.
15 Sukonick DL, Pollock BG, Sweet RA, Mulsant BH, Rosen J, Klunk WE, Kastango KB, DeKosky ST, Ferrell RE: The
5-HTTPR *S/*L polymorphism and aggressive behaviour in Alzheimer’s disease. Arch Neurol 2001;58:1425–
1428.
16 Sweet RA, Pollock BG, Sukonick DL, Mulsant BH, Rosen J, Klunk WE, Kastango KB, DeKosky ST, Ferrell RE: The
5-HTTPR polymorphism confers liability to a combined phenotype of psychotic and aggressive behavior in
Alzheimer disease. Int Psychogeriatr 2001;13:401–409.
17 Borroni B, Grassi M, Agosti C, Archetti S, Costanzi C, Cornali C, Caltagirone C, Caimi L, Di Luca M, Padovani A:
Cumulative effect of COMT and 5-HTTLPR polymorphisms and their interaction with disease severity and
comorbidities on the risk of psychosis in Alzheimer disease. Am J Geriatr Psychiatry 2006;14:343–351.
18 Pitchard AL, Pritchard CW, Bentham P, Lendon CL: Role of serotonin transporter polymorphisms in the behav-
ioural and psychological symptoms in probable Alzheimer disease patients. Dement Geriatr Cogn Disord
2007;24:201–206.
19 Quaranta D, Bizzarro A, Marra C, Vita MG, Seripa D, Pilotto A, Sebastiani V, Mecocci P, Masullo C: Psychotic
symptoms in Alzheimer’s disease and 5-HTTLPR polymorphism of the serotonin transporter gene: evidence
for an association. J Alzheimers Dis 2009;16:173–180.
20 Proitsi P, Lupton MK, Reeves SJ, Hamilton G, Archer N, Martin BM, Iyegbe C, Hollingworth P, Lawlor B, Gill M,
Brayne C, Rubinsztein DC, Owen MJ, Williams J, Lovestone S, Powell JF: Association of serotonin and dopamine
gene pathways with behavioral subphenotypes in dementia. Neurobiol Aging 2012;33:791–803.
21 Li T, Holmes C, Sham PC, Vallada H, Birkett J, Kirov G, Lesch KP, Powell J, Lovestone S, Collier D: Allelic func-
tional variation of serotonin transporter expression is a susceptibility factor for late onset Alzheimer’s disease.
Neuroreport 1997;8:683–686.
22 Rocchi A, Micheli D, Ceravolo R, Manca ML, Tognoni G, Siciliano G, Murri L: Serotoninergic polymorphisms
(5-HTTLPR and 5-HT2A): association studies with psychosis in Alzheimer disease. Genet Test 2003;7: 309–
314.
23 Assal F, Alarcón M, Solomon EC, Masterman D, Geschwind DH, Cummings JL: Association of the serotonin
transporter and receptor gene polymorphisms in neuropsychiatric symptoms in Alzheimer disease. Arch
Neurol 2004;61:1249–1253.
24 Ha TM, Cho DM, Park SW, Joo MJ, Lee BJ, Kong BG, Kim JM, Yoon JS, Kim YH: Evaluating associations between
5-HTTLPR polymorphism and Alzheimer’s disease for Korean patients. Dement Geriatr Cogn Disord 2005;20:
31–34.
25 Micheli D, Bonvicini C, Rocchi A, Ceravolo R, Mancuso M, Tognoni G, Gennarelli M, Siciliano G, Murri L: No
evidence for allelic association of serotonin 2A receptor and transporter gene polymorphisms with depression
in Alzheimer disease. J Alzheimers Dis 2006;10:371–378.
Vanderbilt University - Eskind Biomedical Library

26 Ueki A, Ueno H, Sato N, Shinjo H, Morita Y: Serotonin transporter gene polymorphism and BPSD in mild
Alzheimer’s disease. J Alzheimers Dis 2007;12:245–253.
129.59.95.115 - 9/13/2016 3:32:57 AM

27 Grünblatt E, Zehetmayer S, Bartl J, Löffler C, Wichart I, Rainer MK, Jungwirth S, Bauer P, Danielczyk W, Tragl
KH, Riederer P, Fischer P: Genetic risk factors and markers for Alzheimer’s disease and/or depression in the
VITA study. J Psychiatr Res 2009;43:298–308.
Downloaded by:
 Dement Geriatr Cogn Disord 2016;41:334–347 346
DOI: 10.1159/000447324 © 2016 S. Karger AG, Basel
www.karger.com/dem
Yamazaki et al.: Polymorphisms, Methylation Profiles, and Peripheral mRNA Expression
of the Serotonin Transporter Gene in Patients with Alzheimer’s Disease

28 Albani D, Prato F, Tettamanti M, Lovati C, Galimberti D, Restelli I, Mariani C, Quadri PL, Scarpini E, Lucca U,
Forloni G.: The serotonin transporter promoter polymorphic region is not a risk factor for Alzheimer’s disease
related behavioral disturbances. J Alzheimers Dis 2009;18:125–130.
29 Iga J, Ueno S, Yamauchi K, Motoki I, Tayoshi S, Ohta K, Song H, Morita K, Rokutan K, Ohmori T: Serotonin trans-
porter mRNA expression in peripheral leukocytes of patients with major depression before and after treatment
with paroxetine. Neurosci Lett 2005;389:12–16.
30 Tsao CW, Lin YS, Chen CC, Bai CH, Wu SR: Cytokines and serotonin transporter in patients with major
depression. Prog Neuropsychopharmacol Biol Psychiatry 2006;30:899–905.
31 Thomas AJ, Hendriksen M, Piggott M, Ferrier IN, Perry E, Ince P, O’Brien JT: A study of the serotonin trans-
porter in the prefrontal cortex in late-life depression and Alzheimer’s disease with and without depression.
Neuropathol Appl Neurobiol 2006;32:296–303.
32 Chen KH, Reese EA, Kim HW, Rapoport SI, Rao JS: Disturbed neurotransmitter transporter expression in
Alzheimer’s disease brain. J Alzheimers Dis 2011;26:755–766.
33 Creese B, Ballard C, Jones E: Cognitive impairment in studies of 5HTTLPR and psychosis in Alzheimer’s disease:
a systematic review. Dement Geriatr Cogn Disord 2013;35:155–164.
34 Watanabe SY, Iga J, Numata S, Umehara H, Nishi A, Kinoshita M, Inoshita M, Ohmori T: Polymorphism in the
promoter of the gene for the serotonin transporter affects the age of onset of major depressive disorder in the
Japanese population. J Affect Disord 2015;183:156–158.
35 Ouchi Y, Yoshikawa E, Futatsubashi M, Yagi S, Ueki T, Nakamura K: Altered brain serotonin transporter and
associated glucose metabolism in Alzheimer disease. J Nucl Med 2009;50:1260–1266.
36 McKhann GM, Knopman DS, Chertkow H, Hyman BT, Jack CR Jr, Kawas CH, Klunk WE, Koroshetz WJ, Manly JJ,
Mayeux R, Mohs RC, Morris JC, Rossor MN, Scheltens P, Carrillo MC, Thies B, Weintraub S, Phelps CH: The diag-
nosis of dementia due to Alzheimer’s disease: recommendations from the National Institute on Aging-
Alzheimer’s Association workgroups on diagnostic guidelines for Alzheimer’s disease. Alzheimers Dement
2011;7:263–269.
37 Folstein MF, Folstein SE, McHugh PR: ‘Mini-mental state’: a practical method for grading the cognitive state of
patients for the clinician. J Psychiatr Res 1975;12:189–198.
38 Morris JC: The Clinical Dementia Rating (CDR): current version and scoring rules. Neurology 1993;43:2412–
2414.
39 Cummings JL, Mega M, Gray K, Rosenberg-Thompson S, Carusi DA, Gornbein J: The Neuropsychiatric Inventory:
comprehensive assessment of psychopathology in dementia. Neurology 1994;44:2308–2314.
40 De Luca V, Tharmalingam S, King N, Strauss J, Bulgin N, Kennedy JL: Association study of a novel functional
polymorphism of the serotonin transporter gene in bipolar disorder and suicidal behavior. Psychopharma-
cology (Berl) 2005;182:128–131.
41 De Luca V, Zai G, Tharmalingam S, de Bartolomeis A, Wong G, Kennedy JL: Association study between the novel
functional polymorphism of the serotonin transporter gene and suicidal behavior in schizophrenia. Eur
Neuropsychopharmacol 2006;6:268–271.
42 Praschak-Rieder N, Kennedy J, Wilson AA, Hussey D, Boovariwala A, Willeit M, Ginovart N, Tharmalingam S,
Masellis M, Houle S, Meyer JH: Novel 5-HTTLPR allele associates with higher serotonin transporter binding in
putamen: a [(11)C] DASB positron emission tomography study. Biol Psychiatry 2007;62:327–331.
43 Mortensen OV, Thomassen M, Larsen MB, Whittemore SR, Wiborg O: Functional analysis of a novel human
serotonin transporter gene promoter in immortalized raphe cells. Brain Res Mol Brain Res 1999;68:141–148.
44 Carrol ED, Salway F, Pepper SD, Saunders E, Mankhambo LA, Ollier WE, Hart CA, Day P: Successful downstream
application of the Paxgene Blood RNA system from small blood samples in paediatric patients for quantitative
PCR analysis. BMC Immunol 2007;8:20.
45 Hu N, Tan MS, Yu JT, Sun L, Tan L, Wang YL, Jiang T, Tan L: Increased expression of TREM2 in peripheral blood
of Alzheimer’s disease patients. J Alzheimers Dis 2014;38:497–501.
46 Watanabe S, Iga J, Nishi A, Numata S, Kinoshita M, Kikuchi K, Nakataki M, Ohmori T: Microarray analysis of
global gene expression in leukocytes following lithium treatment. Hum Psychopharmacol Clin Exp 2014;29:
190–198.
47 Watanabe SY, Iga J, Ishii K, Numata S, Shimodera S, Fujita H, Ohmori T: Biological tests for major depressive
disorder that involve leukocyte gene expression assays. J Psychiatr Res 2015;66–67:1–6.
48 Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the
2(-Delta Delta C(T)) method. Methods 2001;25:402–408.
49 Kanda Y: Investigation of the freely available easy-to-use software ‘EZR’ for medical statistics. Bone Marrow
Transplant 2013;48:452–458.
50 Bradley SL, Dodelzon K, Sandhu HK, Philibert RA: Relationship of serotonin transporter gene polymorphisms
and haplotypes to mRNA transcription. Am J Med Genet B Neuropsychiatr Genet 2005;136B:58–61.
51 Talati A, Guffanti G, Odgerel Z, Ionita-Laza I, Malm H, Sourander A, Brown AS, Wickramaratne PJ, Gingrich JA,
Weissman MM: Genetic variants within the serotonin transporter associated with familial risk for major
Vanderbilt University - Eskind Biomedical Library

depression. Psychiatry Res 2015;228:170–173.

52 Kunugi H, Ueki A, Otsuka M, Isse K, Hirasawa H, Kato N, Nabika T, Kobayashi S, Nanko S: Alzheimer’s disease
129.59.95.115 - 9/13/2016 3:32:57 AM

and 5-HTTLPR polymorphism of the serotonin transporter gene: no evidence for an association. Am J Med
Genet 2000;96:307–309.
Downloaded by:
 Dement Geriatr Cogn Disord 2016;41:334–347 347
DOI: 10.1159/000447324 © 2016 S. Karger AG, Basel
www.karger.com/dem
Yamazaki et al.: Polymorphisms, Methylation Profiles, and Peripheral mRNA Expression
of the Serotonin Transporter Gene in Patients with Alzheimer’s Disease

53 Tsai SJ, Hong CJ, Liu TY, Cheng CY, Liu HC: Association study for a functional serotonin transporter gene poly-
morphism and late-onset Alzheimer’s disease for Chinese patients. Neuropsychobiology 2001;44:27–30.
54 Fehér A, Juhász A, László A, Pákáski M, Kálmán J, Janka Z: Serotonin transporter and serotonin receptor 2A
gene polymorphisms in Alzheimer’s disease. Neurosci Lett 2013;534:233–236.
55 Lorenzi C, Marcone A, Pirovano A, Marino E, Cordici F, Cerami C, Delmonte D, Cappa SF, Bramanti P, Smeraldi
E: Serotonin transporter and saitohin genes in risk of Alzheimer’s disease and frontotemporal lobar dementia:
preliminary findings. Neurol Sci 2010;31:741–749.
56 Seripa D, Franceschi M, D’Onofrio G, Panza F, Cascavilla L, Paris F, Placentino G, Matera MG, Solfrizzi V, Pilotto
A: Polymorphism C in the serotonin transporter gene (SLC6A4) in questionable dementia and Alzheimer’s
disease. Neurosci Lett 2008;438:335–339.
57 Nishimura AL, Guindalini C, Oliveira JR, Nitrini R, Bahia VS, de Brito-Marques PR, Otto PA, Zatz M: Monoamine
oxidase a polymorphism in Brazilian patients: risk factor for late-onset Alzheimer’s disease? J Mol Neurosci
2005;27:213–217.
58 Zill P, Padberg F, de Jonge S, Hampel H, Bürger K, Stübner S, Boetsch T, Jürgen Möller H, Ackenheil M, Bondy
B: Serotonin transporter (5-HTT) gene polymorphism in psychogeriatric patients. Neurosci Lett 2000; 284:
113–115.
59 Oliveira JR, Gallindo RM, Maia LG, Brito-Marques PR, Otto PA, Passos-Bueno MR, Morais MA Jr, Zatz M: The
short variant of the polymorphism within the promoter region of the serotonin transporter gene is a risk
factor for late onset Alzheimer’s disease. Mol Psychiatry 1998;3:438–441.
60 Porcelli S, Fabbri C, Serretti A: Meta-analysis of serotonin transporter gene promoter polymorphism
(5-HTTLPR) association with antidepressant efficacy. Eur Neuropsychopharmacol 2012;22:239–258.
61 Watanabe SY, Iga J, Ishii K, Numata S, Shimodera S, Fujita H, Ohmori T: Biological tests for major depressive
disorder that involve leukocyte gene expression assays. J Psychiatr Res 2015;66–67:1–6.
62 Martin J, Cleak J, Willis-Owen SAG, Flint J, Shifman S: Mapping regulatory variants for the serotonin trans-
porter gene based on allelic expression imbalance. Mol Psychiatry 2007;12:421–422.
63 Murthy NV, Selvaraj S, Cowen PJ, Bhagwagar Z, Riedel WJ, Peers P, Kennedy JL, Sahakian BJ, Laruelle MA,
Rabiner EA, Grasby PM: Serotonin transporter polymorphisms (SLC6A4 insertion/deletion and rs25531) do
not affect the availability of 5-HTT to [11C] DASB binding in the living human brain. Neuroimage 2010; 52:
50–54.
64 Gao Z, Yuan H, Sun M, Wang Z, He Y, Liu D: The association of serotonin transporter gene polymorphism and
geriatric depression: a meta-analysis. Neurosci Lett 2014;578:148–152.
65 Smits KM, Smits LJ, Schouten JS, Stelma FF, Nelemans P, Prins MH: Influence of SERTPR and STin2 in the sero-
tonin transporter gene on the effect of selective serotonin reuptake inhibitors in depression: a systematic
review. Mol Psychiatry 2004;9:433–441.
66 Goldman N, Glei DA, Lin, YH, Weinstein M: The serotonin transporter polymorphism (5-HTTLPR): allelic vari-
ation and links with depressive symptoms. Depress Anxiety 2010;27:260–269.
67 Murphy DL, Moya PR: Human serotonin transporter gene (SLC6A4) variants: their contributions to under-
standing pharmacogenomic and other functional GxG and GxE differences in health and disease. Curr Opin
Pharmacol 2011;11:3–10.
68 Kim JM, Stewart R, Kang HJ, Kim SW, Shin IS, Kim HR, Shin MG, Kim JT, Park MS, Cho KH, Yoon JS: A longitudinal
study of SLC6A4 DNA promoter methylation and poststroke depression. J Psychiatr Res 2013;47:1222–1227.
69 Domschke K, Tidow N, Schwarte K, Deckert J, Lesch KP, Arolt V, Zwanzger P, Baune BT: Serotonin transporter
gene hypomethylation predicts impaired antidepressant treatment response. Int J Neuropsychopharmacol
2014;17:1167–1176.
70 Devlin AM, Brain U, Austin J, Oberlander TF: Prenatal exposure to maternal depressed mood and the MTHFR
C677T variant affect SLC6A4 methylation in infants at birth. PLoS One 2010;5:e12201.
71 Alasaari JS, Lagus M, Ollila HM, Toivola A, Kivimäki M, Vahtera J, Kronholm E, Härmä M, Puttonen S, Paunio T:
Environmental stress affects DNA methylation of a CpG rich promoter region of serotonin transporter gene in
a nurse cohort. PLoS One 2012;7:e45813.
72 Kang HJ, Kim JM, Stewart R, Kim SY, Bae KY, Kim SW, Shin IS, Shin MG, Yoon JS: Association of SLC6A4 meth-
ylation with early adversity, characteristics and outcomes in depression. Prog Neuropsychopharmacol Biol
Psychiatry 2013;44:23–28.
73 Okada S, Morinobu S, Fuchikami M, Segawa M, Yokomaku K, Kataoka T, Okamoto Y, Yamawaki S, Inoue T,
Kusumi I, Koyama T, Tsuchiyama K, Terao T, Kokubo Y, Mimura M: The potential of SLC6A4 gene methylation
analysis for the diagnosis and treatment of major depression. J Psychiatr Res 2014;53:47–53.
74 Philibert RA, Sandhu H, Hollenbeck N, Gunter T, Adams W, Madan A: The relationship of 5HTT (SLC6A4) meth-
ylation and genotype on mRNA expression and liability to major depression and alcohol dependence in
subjects from the Iowa Adoption Studies. Am J Med Genet B Neuropsychiatr Genet 2008;147B:543–549.
75 Belzeaux R, Azorin JM, Ibrahim EC: Monitoring candidate gene expression variations before, during and after
a first major depressive episode in a 51-year-old man. BMC Psychiatry 2014;14:73.
Vanderbilt University - Eskind Biomedical Library

76 Mori T, Shimada H, Shinotoh H, Hirano S, Eguchi Y, Yamada M, Fukuhara R, Tanimukai S, Zhang MR, Kuwabara
S, Ueno S, Suhara T: Apathy correlates with prefrontal amyloid β deposition in Alzheimer’s disease. J Neurol
129.59.95.115 - 9/13/2016 3:32:57 AM

Neurosurg Psychiatry 2013;85:449–455.

Downloaded by: