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Abstract

Background: stress disturbs one of the key biological processes: sleep.


Previous researches have also shown a strong influence of depression on sleep quality.
However, the effects of single nucleotide polymorphism of CLOCK gene on sleep remains
to be investigated.
Methods: Participants consisted of 580 Chinese university faculty and staff (291
males and 284 females, 5 missing data). Work Stress Scale, Self-rating depression scale
and Pittsburgh Sleep Quality Index were applied to measure the stress, depression and
sleep resepctively. A SNP on CLOCK gene (rs11932595) was tested.
Results: The mediation effect of depression on the relationship between stress and PSQI
is significant. rs11932595 shows a significant main effect in the interaction between stress
and PSQI.
Limitation: We used self-reported questionnaire to generate our data, further
experimentally controlled study should be performed to verify our results.
Conclusion: Our findings demonstrated that depression influences the relation between
stress and sleep quality. Our findings also provide genetic evidence for job-related stress
and sleep disturbance, indicating the popular fact that highly stressed working population
is more likely to have sleep problems could be likely to trace back to the genetic variants.

Introduction
One of the most important regulators of daily activities is the circadian rhythm, which is
controlled by the CLOCK system. Light information is sent from the retina to suprachiasmatic
nuclei (SCN),the central CLOCK system. Neurons of the central CLOCK in the SCN send
efferent projections to the other parts of the brain, such as the paraventricular nucleus (PVN),
and also uses humoral mediators, such as AVP, to synchronize circadian rhythmicity of
peripheral CLOCK systems outside of the brain in order to regulate body functions (Nicolaides
et al,2014; Nader et al, 2010). The molecular mechanism behind this regulation is
CLOCK/BMAL heterodimer can activate transcription of Per and Cry genes, while PER and
CRY protein provide negative feedback on CLOCK/BMAL activities (Ko et al, 2006). Besides
the regular circadian rhythm, human and other animals also have to face various stressors
throughout the life. Stressors mainly activate hypothalamus-pituitary-adrenal(HPA) axis, which
produces glucocorticoid as its end effector. glucocorticoid exerts its effect by binding to
glucocorticoid receptors, which then binds to promotor elements of target genes in order to
regulate their transcription(Nicolaides et al,2014). The central CLOCK system modulates
glucocorticoid release from the adrenal glands by influencing the activity of the HPA axis
through efferent connections from the SCN to the CRH/AVP-containing neurons of the PVN or
by HPA axis-independent fashion (Nader et al, 2010)However, the central CLOCK system
remains unaffected and standardizes the peripheral CLOCKs back to normal when stress
has passed.

Sleep is an important part of daily activities for almost all animal species as it provides time for
restoration and growth of many organ systems. Therefore, it is critical that sleep occurs in
appropriate time and in sufficient length for such purposes. Sleep is therefore tightly regulated
precisely for this reason. Sleep regulation is conducted primarily through two mechanisms:
homeostatic regulation and circadian regulation.The homeostatic mechanism balances sleep
need accumulated during wake time and the corresponding release of such need during sleep,
while circadian regulation places sleep in an appropriate time frame (Cirelli,2009; Landgraf et
al, 2012). Therefore, sleep is significantly affected by damages of the circadian system. Indeed,
both SCN lesions and mutations of circadian genes have been shown to result in sleep
disorders in both animal models and human (Tafti, 2009; Sehgal, 2011). As described in the
previous paragraph, stress can cause HPA axis to release glucocorticoid which perturbs normal
circadian rhythm. Such perturbance unavoidably lead to change in sleep patterns and
constitutes a major cause of sleep disorders. Disturbed circadian regulation is not the only
reason why stress can negatively affect sleep quality and quantity. By affecting the homeostatic
regulation of sleep, different kinds of stress hormones released during stressful events also
have tremendous impact on sleep. Unsurprisingly, a relatively low level of stress hormones is
required for sleep, as the role of stress hormones is to prepare animals for stressful situations
which is incompatible with sleep. Increased level of stress hormones, such as cortisol, have
been shown to shorten sleep duration and disturb normal sleep pattern.(Suchecki et al, 2012)
Dysregulation of HPA activity is associated with various sleep disorders (Buckley et al, 2005).
From these evidences, it is clear that stress disturbs sleep through inappropriate timing of HPA
activation.

Multiple studies have shown that various aspects of job stress are strongly correlated with and
predicts the onset of sleep disturbance. For example, work ruminaiton has been shown to
correlate with sleep problem significantly(Cropley et al, 2006). Moreover, a prospective study
done by Linton among Swedish workers showed that those under significant stress are twice
as likely to have sleep-related disorders (Linton, 2004). Finally, another study among American
workers showed that increase in stress leads to increase in all three types of poor sleep quality:
difficulty initiating sleep, difficulty maintaining sleep and non-restorative sleep (Knudsen et al,
2007). These studies and others have all pointed out the strong effect of stress on sleep
disturbance.

In addition to molecular mechanisms underlying the relationship between stress and sleep,
multiple studies have validated that job stress can negatively influence sleep quality. These
studies are conducted in different countries using people of different occupation (Takaki et al,
2010; Linton, 2004; Cropley et al, 2006; Knudsen et al, 2007; Song et al, 2017). Therefore,
stress induced sleep disturbance is clearly a global phenomenon common to people with
diverse cultural and ethnical background. Besides job stress, depression have also been
indicated as a major contributor of poor sleep quality(Hall et al, 2000; Nutt, 2008). Since there
is a well-established connection between stress and depression (Hammen, 2005), we propose
that depression may play an interactive role between stress and sleep. Genetic variation in the
circadian rhythm regulation constitutes another important influence of sleep quality (Allebrandt,
2010). Specifically, single nucleotide polymorphisms(SNPs) of circadian genes have been
investigated for the effects in sleep regulation. Therefore, our second hypothesis is that SNPs
on the CLOCK gene can mediate the relationship between stress and sleep.

Method
Participants
The subjects of this study were recruited from faculty and staff members of an university in
Beijing. They were selected based on 2 criteria:first, they must be currently employed by the
university, second, they do not have any past history of mental or psychiatric disorders.
Informed consent were obtained from all participants in written form. Out of the 591 responses
received, 580 valid subjects were selected based on the above criteria. This study was
approved by the Ethnic Committee of Peking University.

Measures
Demographical Data
Participants completed a basic questionnaire comprising of questions on gender, age,
educational background, job title and position, physical and mental conditions.
Job Stress
Job Stress was measured using the Work Stress Scale developed by House and Rizzo,
translated into Chinese by Ma and Liang. (House and Rizzo, 1972; Ma and Liang, 1997) This
questionnaire is composed of 11 items such as “I often feel worried about my job” and “My work
is a heavy mental burden.” Responses to each item is rated on a scale of 1-6, 1 being
completely disagree and 6 being completely agree. Therefore, the total score ranges from 11-
66, with higher score corresponds to higher level of stress.
Depression
The Chinese version of Zung’s Self-rating depression scale (SDS) was used for the
present study (Zung, 1965). The questionnaire consists of 20 questions, each on a scale of 1-
4. Raw score is the sum of each individual rating. The standard score is then calculated by
multiplying the raw score with 1.25.
Sleep quality
Sleep disturbance was measured by Pittsburgh Sleep Quality Index (PSQI), a self-
reported questionnaire developed by Buysse (Buysse et al, 1989). The Mandarin Chinese
version was translated and its reliability and validity was verified in a study in 2005 (Tsai et al,
2005). This questionnaire assesses sleep quality of the previous month using seven
components: subjective sleep quality, sleep latency, sleep duration, habitual sleep efficiency,
sleep disturbances, use of sleeping medication, and daytime dysfunction. Each component
generates a score from 0 to 3. The total score is the sum of each individual components. Lower
score indicates a better overall sleep quality.

DNA extraction and SNP polymorphism testing procedures.


5ml venous blood sample was taken from each subject at morning fasting state. White blood
cell DNA is precipitated and purified from cell lysate. The genotype of SNP polymorphism is
then identified using polymerase chain reaction(PCR). The full sequence of primers used in
PCR is presented in Supplementary Table 1

Statistical Analysis
All data analysis were done using SPSS24.0. Data on stress, depression and sleep quality are
presented as mean ± standard deviation. Pearson regression was used to calculate the
correlation coefficient between stress, depression and each component of PSQI. Regression
analysis was also used to determine the interaction effect between stress, depression and sleep
quality. Mediation effect of SNP genotype on stress and sleep quality was assessed using single
variable general linear model. Independent sample T-test was employed to examine the
difference of each component of PSQI between different SNP genotypes. Significance level of
0.05 was set for all hypothesis tests performed.

Results
Demographical data of participants collected from initial questionnaire are displayed in Table
1. Out of the 580 individuals surveyed, there were 291 male participants (50.2%). and the
mean age (± standard deviation) was 39.70 ± 9.493 years. Out of all the participants,
197(34.0%)has a Ph. D degree, 243(41.9%) has a master degree, 108(18.6%) has a
Bachelor degree, and 21(3.6%) has a degree below the bachelor’s.

Characterization of stress, depression, PSQI scores according to age, gender and education
level are presented in Table 2. The mean score for stress, depression, PSQI are 32.11 ±
10.592, 34.36 ± 8.147, 4.99 ± 2.845, respectively. Difference among these scores is analyzed
using t-test for gender and ANOVA for age and educational level. The mean score for stress
was lower in females than in males(p=0.023).No gender differences were found in relation to
the depression and PSQI scores. Both stress and PSQI score difference are significant
across different age groups(p=0.001 for PSQI and p=0.02 for stress). Depression score is
significant among educational level.

Regression coefficients between stress, depression, total score of PSQI and each component
of PSQI are shown in Table 3. We found that stress, depression and PSQI are significantly
correlated with each other (r^2=0.483, p<0.001 between stress and depression, r^2=0.393,
p< 0.001 between stress and PSQI, r^2=0.432, p<0.001 between depression and PSQI)
Stress and depression are also significantly correlated with most components of PSQI.

Results from mediation analysis of stress and PSQI with depression being the mediator are
shown in Table 4 and Figure 1. Both the total (beta=0.1053, p<0.001) and direct
effect(beta=0.064, p<0.001) of stress on PSQI are positive and significant. Effect of stress on
depression (beta=0.361, p<0.001) and effect of depression on PSQI (beta=0.1142,p<0.001)
are also positive and significant.

The interaction effect of CLOCK gene polymorphism between stress and PSQI is shown in
Table 5 and Figure 2. While SNP variation of CLOCK gene at locus rs11932595 shows a
significant main effect on PSQI(F=6.407, p=0.012), the interaction with stress failed to be
significant(F=0.350, p=0.705). The population was divided into low, medium and high stress
groups using one standard deviation above and below the mean as criteria. The graph shows
a linear relationship between PSQI and stress level for the population with AA genotype.
However, there is a dramatic increase in PSQI score for participants with GG or GA genotype
at high stress level.

Independent sample t-test of PSQI and each component of PSQI between 2 groups of SNP
variation are displayed in Table 6. Total PSQI scores are significant between the 2 groups but
the individual components are not, except for sleep latency and sleep duration.

Independent sample t-tests of stress, depression, PSQI between 2 groups of SNP variation
for male and female population are displayed in Table 7. Total PSQI score is significant
between the 2 groups for the male population while stress score is significant for female
population.

Discussion

This study identified the relationships between stress, depression, SNP polymorphism and
sleep quality using SDS, PSQI and genotyping methods among Chinese local university
faculty and administrative staff. The result of the present study indicates the relationship
between job stress, depression and poor sleep quality. While the effect of both stress and
genotype on sleep have been investigated, there are few studies which considered the
genetic and environmental influences at the same time in order to draw a more
comprehensive conclusion.

The main findings of this study are as below. First, there is significant gender difference of
stress. Individuals aged from 30-39 has the lowest sleep quality and is likely the result of high
stress level. We also found education level is an influencer of depression. The three aspects
of this study: stress, depression and sleep are strongly correlated with each other. Depression
is a mediator between job stress and PSQI score. People with homozygous A on the CLOCK
gene polymorphism site rs11932595 have better sleep quality than people carrying G allele.

A study done on Chinese clinical nurses found that female nurses have a significantly higher
PSQI scores than male nurses, which is inconsistent with our results (Dong et al, 2017). This
inconsistency could be due to different occupations surveyed( nurses vs university faculty and
staff) among other reasons. Another longitudinal three-wave study on Swedish working
population showed that chronically high job demand is a risk factor for low sleep quality (Van
Laethem, 2018). This result agrees with our study since job demand often lead to job stress.

We found significant positive correlation between stress, sleep and depression. In another
study in which a single item stress question (SISQ) was used to measure work-related stress,
it predicted sick leave, depression and exhaustion at 12 month follow-up. In particular, there is
significant positive correlation between SISQ and depression (Arapovic-Johansson et al,
2017). Also consistent with our study, a cross-sectional study performed on korean dentists
showed PSQI score is significantly associated with job stress and depression. It also reported
an average PSQI score of 5.26± 2.96, close to our result( Song et al. 2017)

Consistent with our result, a study done on undergraduate students showed that a higher
number of G alleles in CLOCK gene variation rs11932595 is associated with higher level of
self-reported sleep difficulty, although it is not associated with any objective measure of sleep
(Vanderlind et al, 2014). In a study involving ADHD male children, no significant difference
was found for rs11932595 alleles (Jin et al, 2016). A meta-analysis of South Tyrol and Estonia
population found that homogeneous A genotype of rs11932595 is significantly associated with
long sleep duration (Allebrandt et al, 2010).

We found that depression mediated the relationship between stress and sleep. In a study
done in elderly Chinese population, the authors found that short sleep duration and poor
sleep quality are inversely related to life satisfaction and such relationship is partially
mediated by depression. (Zhi et al, 2016) Similarly another study done on elderly adults
showed that sleep quality acted as a mediator between depression and quality of life (Becker
et al, 2018). A cross sectional study of elderly in urban communities stated that sleep quality
partially mediated the effect of perceived stress on depression (Liu et al, 2017).

We have found that stress and PSQI levels are different among different genders.
For the male population, the PSQI scores showed a significant difference between the 2
CLOCK gene variants(p=.003). For the female population, stress level is significantly different
(p=.012)
The multiple regression analysis between stress, depression and PSQI showed significant
results for almost every pair of variables. Therefore, clearly the instruments we have used are
reliable and valid. However, only a few of these correlation coefficients are high ( .5 or above),
so it seems that a linear model may not be the best in explaining the relationship between
these variables. Our data also shows significant mediating effect of depression between sleep
and PSQI since all path coefficients(beta) are signifiant and the direct effect decreased
significantly from the total effect. Our interaction analysis of SNP polymorphism of CLOCK
gene showed that population with G/GA genotype has particularly high PSQI score when
subjected under high stress. This result is in agreement with our previous findings.

Our present study have several limitations. First, we used self-reported questionnaires to
generate data for stress, depression and sleep quality, which means that our scale is
subjective. The causal relationship between the variables studied is also difficult to identify
since we performed a cross-sectional study rather than longitudinal study. As previously
mentioned, our regression analysis did not generate large correlation coefficient, which
means that the relationship between these variables are explained poorly by a linear model.
Further study is necessary in order to identify appropriate regression model for analysis. Also,
we should have separated our population according to gender since male and female
participants have different level of stress.

Despite these limitations, we propose a model in which the relationship between stress and
sleep quality is partially mediated by depression. Therefore our result suggests reducing
depression is a good way of maintaining good sleep status. In addition, a SNP within the
CLOCK gene was associated with sleep quality. We believe our proposed model and SNP
analysis are an excellent starting point for the development of interdisciplinary models of
sleep disturbance.

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