Association of serum BDNF levels and the BDNF Val66Met polymorphism with the sleep pattern in healthy young adults

Abstract

Background
Brain-derived neurotrophic factor (BDNF) is widely expressed in the brain and plays an important role in neuronal maintenance, plasticity, and neurogenesis. Prior studies have found that decreased serum BDNF levels are associated with perceived stress, depression, or sleep disturbances in humans.

Study objectives
To elucidate whether the serum BDNF levels and BDNF genotype were associated with the sleep pattern in healthy young adults.

Methods
The study group consisted of 79 healthy paid volunteers (45 men, 34 women) aged 20 to 29 years. Serum BDNF levels were measured with an enzyme-linked immunosorbent assay, and a single-nucleotide polymorphism (Val66Met) in the BDNF gene was assessed with a TaqMan assay. Details of the sleep pattern were obtained from 1-week sleep/wake records.

Results
Serum BDNF levels were significantly associated with sleep parameters on weekends, whereas no such association was found on weekdays. On weekends, longer total sleep time and time in bed, and later mid-sleep time were associated with lower serum BDNF levels. The difference between mid-sleep time on weekdays and that on weekends, otherwise known as social jetlag, was negatively associated with serum BDNF levels. Met/Met homozygotes of the BDNF Val66Met polymorphism had significantly longer time in bed on weekends than Val/Val homozygotes. Heterozygotes did not differ from Val/Val homozygotes.

Conclusions
We first found that serum BDNF levels and the BDNF Val66Met polymorphism in healthy young adults were associated with the sleep pattern on weekends but not with that on weekdays, suggesting that the systems involved in BDNF control may be linked to endogenous sleep characteristics rather than the socially constrained sleep schedule in healthy young adults.

Full text

RESEARCH ARTICLE
Association of serum BDNF levels and the
BDNF Val66Met polymorphism with the sleep
pattern in healthy young adults
Kaori Saitoh
☯
, Ryuji Furihata
☯
, Yoshiyuki Kaneko, Masahiro Suzuki, Sakae Takahashi,
Makoto Uchiyama*
Department of Psychiatry, Nihon University School of Medicine, Tokyo, Japan
☯These authors contributed equally to this work.
*suzuki.masahiro94@nihon-u.ac.jp
Abstract
Background
Brain-derived neurotrophic factor (BDNF) is widely expressed in the brain and plays an
important role in neuronal maintenance, plasticity, and neurogenesis. Prior studies have
found that decreased serum BDNF levels are associated with perceived stress, depression,
or sleep disturbances in humans.
Study objectives
To elucidate whether the serum BDNF levels and BDNF genotype were associated with the
sleep pattern in healthy young adults.
Methods
The study group consisted of 79 healthy paid volunteers (45 men, 34 women) aged 20 to 29
years. Serum BDNF levels were measured with an enzyme-linked immunosorbent assay,
and a single-nucleotide polymorphism (Val66Met) in the BDNF gene was assessed with a
TaqMan assay. Details of the sleep pattern were obtained from 1-week sleep/wake records.
Results
Serum BDNF levels were significantly associated with sleep parameters on weekends,
whereas no such association was found on weekdays. On weekends, longer total sleep
time and time in bed, and later mid-sleep time were associated with lower serum BDNF
levels. The difference between mid-sleep time on weekdays and that on weekends, other-
wise known as social jetlag, was negatively associated with serum BDNF levels. Met/Met
homozygotes of the BDNF Val66Met polymorphism had significantly longer time in bed
on weekends than Val/Val homozygotes. Heterozygotes did not differ from Val/Val
homozygotes.
PLOS ONE | https://doi.org/10.1371/journal.pone.0199765 June 26, 2018 1 / 11
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OPEN ACCESS
Citation: Saitoh K, Furihata R, Kaneko Y, Suzuki M,
Takahashi S, Uchiyama M (2018) Association of
serum BDNF levels and the BDNF Val66Met
polymorphism with the sleep pattern in healthy
young adults. PLoS ONE 13(6): e0199765. https://
doi.org/10.1371/journal.pone.0199765
Editor: Kenji Hashimoto, Chiba Daigaku, JAPAN
Received: April 10, 2018
Accepted: June 13, 2018
Published: June 26, 2018
Copyright: ©2018 Saitoh et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data are
within the paper.
Funding: This study was supported in part by
Health Science Research Grants from the Ministry
of Health, Labor and Welfare of the Japanese
Government and by a Research Grant from the
Japan Society for Promoting Science and
Technology Agency.
Competing interests: The authors have declared
that no competing interests exist.

Conclusions
We first found that serum BDNF levels and the BDNF Val66Met polymorphism in healthy
young adults were associated with the sleep pattern on weekends but not with that on week-
days, suggesting that the systems involved in BDNF control may be linked to endogenous
sleep characteristics rather than the socially constrained sleep schedule in healthy young
adults.
Introduction
Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, is widely
expressed in the brain and the periphery, and plays an important role in neuronal mainte-
nance, plasticity, and neurogenesis [1,2]. Circulating levels of BDNF have been proposed as a
possible marker of diseases [3–5]. BDNF levels are lower in humans with depression [6,7],
bipolar disorder [8,9], schizophrenia [10,11], and dementia [12] compared to age-matched
controls. In addition, several studies demonstrated that successful treatment of depression [13]
and bipolar disorder [14] normalizes peripheral BDNF concentrations, suggesting that BDNF
is a possible marker of disease recovery. Studies on non-clinical populations demonstrated
that perceived stress is negatively associated with circulating BDNF levels. A negative correla-
tion is present between serum BDNF levels and work-related perceived stress in workers [15,
16], and romantic stress in healthy young adults [17]. Serum BDNF levels are also positively
associated with stress resilience in healthy women [18]. Several reports have documented that
decreased circulating BDNF levels are associated with disturbed sleep [19,20]. An epidemio-
logical survey revealed that serum BDNF levels are lower in women with sleep disturbances
[19]. Other studies have reported that serum BDNF levels are correlated with perceived sever-
ity of insomnia [20], and polysomnographically confirmed disturbances in non-rapid eye
movement sleep [21], i.e., reduced theta EEG activity and reduced percentage of deep non-
rapid eye movement sleep. These studies imply that BDNF may participate in homeostatic reg-
ulation of sleep that controls brain recovery.
These previous studies have shown that circulating BDNF may be a state-dependent marker
in patients with psychiatric disorders, an objective marker of psychological stress in healthy
subjects, and a potential indicator of insufficient sleep and a consequence of poor brain recov-
ery in healthy subjects. More recently, an observational study from Switzerland examined the
relationship among stress, sleep, and BDNF levels and suggested an interaction between
BDNF levels and sleep [22], which plays an essential role in brain recovery. This relationship
may also provide an explanation for decreased BDNF levels in subjects complaining of per-
ceived stress and levels in patients with stress-related mental disorders, because sleep is com-
monly disturbed both in stressful conditions and most mental disorders [23]. However, a
fundamental understanding of the physiological interaction between BDNF levels and sleep in
healthy humans remains limited, although a relationship between BDNF levels and several
types of sleep pathology have been reported. Here we studied for the first time the relationship
between the sleep pattern and serum BDNF levels, and between the sleep pattern and BDNF
genotypes in healthy young subjects. Based on previous findings of an age-related decline in
BDNF levels [24,25], we employed healthy young adults within a narrow age range (20 to 40
years) to exclude the confounding effects of age in the present study.
BDNF and the sleep pattern
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Methods
Study subjects and data collection
Subjects were recruited via flyers posted on the Nihon University bulletin board from July
2015 to October 2015. A total of 103 individuals applied to participate in the study. Partici-
pants were selected by psychiatric specialists (KS and MU) using the following criteria: 1)
those aged 20 to 40 years, 2) those who did not engage in shift or midnight work, 3) those who
were free from social dysfunction, 4) those who did not have a history of sleep, neurological, or
psychiatric disorders, or any history of using psychoactive drugs, and 5) those whose score on
the Japanese version of the Center for Epidemiologic Studies Depression Scale [26] was less
than 16 points.
Finally, we recruited 79 healthy paid volunteers (45 men and 34 women) aged 20 to 29
years in the study, and excluded 24 for the following reasons: age >40 years (n = 5); shift work
(n = 5); social dysfunction (n = 1); psychoactive drugs (n = 2); Center for Epidemiologic Stud-
ies Depression Scale scores 16 (n = 16). The participants were medical students or doctors,
and no one had type 2 diabetes, which potentially influences serum BDNF levels [27].
The study was comprised of three parts: (1) evaluation of the sleep pattern based on sleep/
wake records and a questionnaire, (2) measurement of serum BDNF concentration, and (3)
genotyping of a BDNF polymorphism. All the sleep data and blood samples were obtained
within 10 days. The study was approved by the local ethics committee of Nihon University
School of Medicine. Written informed consent was obtained from all individuals who applied
to participate in the study.
Sleep/Wake records. The participants were instructed to record 1) bed time, 2) wake-up
time, and 3) sleep onset latency every day for 7 days or more. Sleep onset time, time in bed
(TIB), total sleep time (TST), and mid-sleep time (MS) were calculated from 1)-3) and were
averaged separately for weekdays and weekends. We further calculated social jetlag as defined
below.
Sleep onset time: bed time −sleep onset latency
TIB: time from bed time to wake-up time
TST: time from sleep onset time to wake-up time
MS: mid-point of sleep onset time and wake-up time
Social jetlag: mean MS of weekends–mean MS of weekdays
Questionnaire. We obtained the participant’s body weight, height, alcohol consumption,
and smoking habits from self-reported information. We defined subjects who had smoked
more than 100 cigarettes for over 6 months as habitual smokers, and those who drank over 22
g pure alcohol for more than three times a week as habitual drinkers.
Sleep quality and sleep problems were measured using the Japanese version of the Pitts-
burgh Sleep Quality Index (PSQI-J) [28]. PSQI assesses subjective sleep quality, sleep latency,
sleep duration, habitual sleep efficiency, sleep disturbances, use of sleeping medication, and
daytime dysfunction over the course of 1 month. Subjects with higher total scores have more
serious sleep disorders.
Blood investigations. Serum BDNF examination. We collected blood between 7:00 and
8:30 AM before breakfast and centrifuged the samples, which were stored at −80˚C. We mea-
sured the BDNF concentration with an enzyme-linked immunosorbent assay using a Quanti-
kine1ELISA kit (R&D Systems, Minneapolis, MN, USA).
BDNF polymorphism. Genomic DNA was extracted from peripheral blood leukocytes
using the Genomic DNA extraction kit (TALENT, Trieste, Italy). Genotyping of the single-
nucleotide polymorphism was performed using TaqMan technology on the Applied Biosys-
tems 7500 Fast Real-Time PCR system (Applied Biosystems, Foster City, CA, USA).
BDNF and the sleep pattern
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Statistical analyses
The presence of gender difference was examined with respect to age, body mass index (BMI),
smoking, alcohol, parameters measured in the sleep/wake records, and serum BDNF with the
t-test and χ
2
test. We first examined the distribution of the serum BDNF data with the Kolmo-
gorov-Smirnov test, and the Pearson test was performed to assess the correlations between
serum BDNF and other factors (age, BMI, smoking, alcohol, and parameters measured in the
sleep/wake records). The associations between the Val66Met polymorphism and sleep pattern
were examined with the t-test. The level of significance was set at p <0.05. All statistical analy-
ses were conducted with SPSS ver. 21.0.
Results
Demographic features, serum BDNF levels, and sleep variables of the participants are shown
in Table 1. The Kolmogorov-Smirnov test revealed that serum BDNF levels were normally dis-
tributed. Allele distributions followed Hardy-Weinberg equilibrium (χ
2
= 2.58; p = 0.89).
Genotype frequencies of 78 subjects were Val/Val 0.36 (29/78), Val/Met 0.45 (35/78), and Met/
Met 0.18 (14/78). We did not find any significant differences in age (F = 0.1, p = 0.90) or sex
(χ
2
= 1.29, p = 0.53) among the three genotype groups.
The statistical comparison of wake up time, sleep onset time, TST, TIB, and MST with
respect to weekdays and weekends is shown in Table 2. These sleep parameters differed signifi-
cantly between weekdays and weekends.
Correlation analyses between serum BDNF levels and age, BMI, smoking habits, alcohol
habits, and the PSQI score did not reveal any significant differences (Table 3). Serum BDNF
levels were not correlated with any sleep parameters on weekdays, whereas the levels were sig-
nificantly correlated with TIB on weekends (r = −0.30, p <0.01), TST on weekends (r = −0.32,
p<0.01), MS on weekends (r = −0.33, p <0.01), and social jetlag (r = −0.28, p <0.05) (Fig 1).
Table 1. Characteristics of study participants.
average SD
Age (years) 24 1.94
Sex (M:F) 45:34
BMI 21.54 3.07
Smoking 8.9%
Drinking alcoholic beverages 27.8%
Serum BDNF (pg/mL) 26060 5814
【Sleep variables】
PSQI 5.09 2.34
Wake-up time weekdays (h:min) 7:22 1:03
Wake-up time weekends (h:min) 8:16 1:39
TST weekdays (h) 6.42 1.38
TST weekends (h) 6.94 1.17
TIB weekdays (h) 6.70 1.35
TIB weekends (h) 7.16 1.17
MS weekdays (h:min) 3:26 0:48
MS weekends (h:min) 3:40 0:40
Social jetlag (h) 0.22 0.96
SD: standard deviation, BMI: body mass index, PSQI: the Pittsburgh Sleep Quality Index, TST: total sleep time, TIB:
time in bed, MS: mid-sleep time.
https://doi.org/10.1371/journal.pone.0199765.t001
BDNF and the sleep pattern
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Distribution patterns and regression lines of parameters showing a significant correlation are
presented in Fig 1.
In terms of sleep variables either on weekdays or weekends, the Val homozygotes and het-
erozygotes did not differ significantly, nor did Val homozygotes and Met homozygotes except
for TIB on weekends, which was nearly 1 hour shorter in Val homozygotes than in Met homo-
zygotes (Table 4,Fig 2).
Discussion
This is the first study to investigate the relationships of serum BDNF levels and BDNF geno-
type with sleep habits such as chronotype and social jetlag in healthy subjects. The key findings
were 1) serum BDNF levels were significantly correlated with sleep parameters, including TST
on weekends, TIB on weekends, MS on weekends, and social jetlag, and 2) Met/Met homozy-
gotes showed significantly longer time in bed on weekends than Val/Val homozygotes.
Previous reports have pointed out that, in modern society, one’s sleep timing on weekdays
is mainly determined by school, work, and/or social constraints irrespective of the internal cir-
cadian clock, whereas on weekends, fewer constraints permit one to sleep on a more preferable
timing according to the internal circadian clock [29,30]. Therefore, sleep timing on weekends
Table 2. The differences in sleep parameters between weekdays and weekends.
Weekdays Weekends p value
Sleep onset time (h:min) 0:57 1:20 0.041
TIB (h) 6.70 7.16 0.015
TST (h) 6.42 6.94 0.007
MS (h:min) 3:26 3:40 0.046
Wake-up time (h:min) 7:22 8:16 0.00002
TST: total sleep time, TIB: time in bed, MS: mid-sleep time.
https://doi.org/10.1371/journal.pone.0199765.t002
Table 3. Serum BDNF levels correlate with the sleep pattern on weekends.
r p-value
Age (years) 0.15 0.19
BMI −0.04 0.71
Smoking 0.80
Drinking alcohol beverages 0.51
PSQI −0.04 0.72
Wake-up time weekdays 0.10 0.37
Wake-up time weekends 0.00 0.99
Sleep onset time weekdays 0.11 0.35
Sleep onset time weekends 0.21 0.06
TST weekdays −0.02 0.84
TST weekends −0.32 0.01
TIB weekdays 0.03 0.82
TIB weekends −0.30 0.01
MS weekdays 0.06 0.58
MS weekends −0.33 0.003
Social jetlag −0.28 0.01
BMI: body mass index, PSQI: the Pittsburgh Sleep Quality Index, TST: total sleep time, TIB: time in bed, MS: mid-
sleep time.
https://doi.org/10.1371/journal.pone.0199765.t003
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or free days has been regarded as a variable that represents the timing of the endogenous circa-
dian rhythm. The MS on weekends, which Zavada et al. [29] first proposed, has been used as a
circadian phase marker that correlates well with differences in chronotype as measured with a
morningness-eveningness questionnaire [31]; early and late MS on weekends indicate morn-
ingness and eveningness, respectively. The present results showing that MS on weekdays was
not correlated with serum BDNF levels but that MS on weekends was negatively correlated
with serum BDNF levels in healthy young adults may indicate that morningness or evening-
ness was associated with increased or decreased serum BDNF levels. These results also provide
the first documentation of the relationship between chronotype and BDNF.
Prior epidemiological surveys have reported that TST on weekends is longer than that on
weekdays in industrialized countries [32,33], and the authors postulated that the discrepancy
is due to sleep insufficiency on weekdays and homeostatic sleep compensation on weekends
Fig 1. Correlations between serum BDNF levels and the sleep pattern on weekends. Analyses showed significant correlations between serum BDNF
levels and the sleep pattern: TST on weekends (r = −0.32, p <0.01), TIB on weekends (r = −0.30, p <0.01), MS on weekends (r = −0.33, p <0.01), and
social jetlag (r = −0.28, p <0.05).
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BDNF and the sleep pattern
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[34–36]. We found that TST and TIB on weekends showed negative correlations with serum
BDNF levels, whereas those on weekdays did not. In the present study, the explanation for
sleep prolongation on weekends seemed to include two possibilities: compensatory sleep pro-
longation for sleep debt on weekdays or natural manifestation of a longer sleep tendency with
fewer social constraints on weekends. Our present results about the relationship between
BDNF and TIB or TST may be interpreted according to these two possibilities.
Social jetlag is defined as the difference between one’s MS on weekdays and that on week-
ends, indicating the degree of sleep misalignment with the endogenous circadian rhythm on
weekdays, which is estimated to account for some psychosomatic difficulties on weekdays,
especially on Monday mornings [37]. We found that social jetlag in the present subjects was
negatively correlated with serum BDNF levels. Given that social jetlag is associated with conse-
quences due to misalignment between the actual sleep schedule and the endogenous circadian
rhythm, BDNF levels may be associated with certain subjective difficulties possibly related to
sleep misalignment [37]. However, we did not find any correlation between serum BDNF lev-
els and the PSQI score. Accordingly, further studies should focus on the relationship between
BDNF and subjective consequences potentially due to such misalignment.
The present analysis of the BDNF Val66Met polymorphism among healthy young subjects
demonstrated that TIB on weekends was significantly longer in Met homozygotes than in Val
homozygotes. A similar non-significant tendency was found in TST on weekends, but no such
differences were found in sleep parameters on weekdays. These results suggest that the BDNF
Val66Met polymorphism had an influence on endogenous sleep characteristics of the subjects.
This is the first documentation of a possible link between the BDNF Val66Met polymorphism
and endogenous sleep characteristics. Other associated features of the BDNF Val66Met poly-
morphism include a potential brain morphological difference in the developmental period
[38]. Hashimoto et al. [38] reported that the volumes of the right cuneus, left insula, and left
ventromedial prefrontal cortex are different between BDNF Met homozygotes and Val homo-
zygotes in adolescents, providing evidence that the Val66Met polymorphism may influence
the volume of the human brain. We postulate that some morphological differences in the
brain affect sleep characteristics. Further studies are needed to examine the relationship.
Table 4. Comparison of the BDNF Val/Val (n = 29), Val/Met (n = 35), and Met/Met (n = 14) genotypes.
Genotype p-value
V/V V/M M/M V/V vs. V/M V/V vs. M/M
PSQI 5.00 5.00 4.50 0.84 0.37
Wake-up time weekdays (h:min) 7:00 7:30 6:54 0.25 0.74
Wake-up time weekends (h:min) 7:45 8:00 8:17 0.48 0.20
Sleep onset time weekdays (h:min) 0:53 1:06 1:07 0.58 0.86
Sleep onset time weekends (h:min) 1:10 1:10 0:42 0.84 0.56
TST weekdays (h) 6.50 6.25 6.50 0.61 0.44
TST weekends (h) 6.82 6.83 7.33 0.62 0.06
TIB weekdays (h) 6.50 6.60 6.67 0.65 0.51
TIB weekends (h) 6.92 7.00 7.71 0.38 0.04
MS weekdays (h:min) 3:23 3:20 3:23 0.91 0.78
MS weekends (h:min) 3:32 3:31 3:43 0.76 0.21
Social jet lag (h) 0.19 0.44 0.43 0.88 0.52
Analyses showed a significant difference in TIB on weekends between Val/Val and Met/Met. V/V: Val/Val, V/M:
Val/Met, M/M: Met/Met, PSQI: the Pittsburgh Sleep Quality Index, TST: total sleep time, TIB: time in bed, MS: mid-
sleep time.
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Mechanisms of the observed relationship between serum BDNF levels and the sleep pattern on
weekends were not clarified in the present study. However, several studies have provided informa-
tion about the mechanisms [39,40]. Chronic sleep loss is associated with increased cortisol levels,
especially around habitual bedtime [39]. An animal study suggested that cortisol affects BDNF lev-
els in the brain [40]. Further study is needed to investigate the role of cortisol in the association
between serum BDNF levels and the sleep pattern observed in the present study.
Limitations
This study has several limitations. First, this was a cross-sectional study, and we could not
determine a causal relationship. A further interventional study is needed to clarify a possible
causal relationship. Second, we measured the sleep pattern with sleep/wake records, which is a
subjective type of assessment. Moreover, we did not evaluate daytime sleep or sleepiness in the
participants. The observations must be confirmed with an objective instrument, such as an
actigraph or polysomnography, and evaluation of daytime sleep or sleepiness, to obtain more
precise results. Third, despite including 79 healthy subjects, this sample size was relatively
small for obtaining a clear relationship between the sleep pattern and serum BDNF levels
including the BDNF polymorphism. Furthermore, we enrolled subjects within a very narrow
age range (20 to 40 years). To increase the generalizability of these findings, future research in
a larger sample size with a wider age range will be required. Finally, we measured serum levels
of BDNF using the Quantikine1ELISA kit (R&D Systems, Minneapolis, MN, USA), which is
commonly used worldwide. This kit measures total BDNF including mature BDNF and its
precursor protein proBDNF. Although mature BDNF is synthesized from proBDNF, recent
studies have reported that proBDNF and mature BDNF elicit opposing effects via the p75
NTR
and TrkM receptors, respectively [41]. Yoshida et al. reported decreased levels of mature
BDNF, but not proBDNF, in major depression [42]. A strong correlation (r = 0.701) was
reported between serum levels of total BDNF using the R&D Systems kit and those of mature
BDNF using the kit from Adipo Bioscience [43]; however, our findings should be confirmed
in future studies by simultaneously measuring mature BDNF levels and proBDNF.
Conclusions
In conclusion, these results suggested that systems involved in BDNF control may be linked to
endogenous sleep characteristics rather than the socially constrained sleep schedule in healthy
young subjects.
Fig 2. A significant difference was found for TIB on weekends between Val homozygotes and Met homozygotes.
https://doi.org/10.1371/journal.pone.0199765.g002
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Acknowledgments
We are grateful to Rei Otsuki, Sakiko Murata, and the students who participated in our study
for their cooperation.
Author Contributions
Conceptualization: Kaori Saitoh, Ryuji Furihata, Yoshiyuki Kaneko, Masahiro Suzuki, Sakae
Takahashi, Makoto Uchiyama.
Data curation: Kaori Saitoh, Ryuji Furihata, Makoto Uchiyama.
Formal analysis: Kaori Saitoh, Ryuji Furihata.
Investigation: Kaori Saitoh, Makoto Uchiyama.
Methodology: Kaori Saitoh, Yoshiyuki Kaneko, Masahiro Suzuki, Sakae Takahashi.
Project administration: Makoto Uchiyama.
Resources: Kaori Saitoh, Sakae Takahashi.
Software: Kaori Saitoh, Ryuji Furihata, Masahiro Suzuki.
Supervision: Masahiro Suzuki, Makoto Uchiyama.
Validation: Ryuji Furihata, Yoshiyuki Kaneko, Masahiro Suzuki, Sakae Takahashi, Makoto
Uchiyama.
Visualization: Kaori Saitoh, Ryuji Furihata.
Writing – original draft: Kaori Saitoh, Ryuji Furihata.
Writing – review & editing: Masahiro Suzuki, Sakae Takahashi, Makoto Uchiyama.
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