Association study of a variable-number tandem repeat polymorphism in the clock gene PERIOD3 and chronotype in Norwegian university students

Circadian rhythms are endogenously generated cycles involving physiological parameters, such as core body temperature, hormone levels, blood pressure, sleep, and metabolism, with a period length of around 24?h. The circadian clock in mammals is regulated by a set of clock genes that are functionally linked together, and polymorphisms in clock genes could be associated with differences in circadian rhythms. A variable-number tandem repeat (VNTR) in the human clock gene PERIOD3 (PER3) has been suggested to correlate with a morning (lark) versus evening (owl) chronotype as well as with the circadian rhythm sleep disorder ?delayed sleep phase disorder? (DSPD). The authors examined 432 healthy Norwegian university students in search of further support for an association between the PER3 polymorphism and diurnal preference. The Horne-?stberg Morningness-Eveningness Questionnaire (MEQ) and Preferences Scale (PS) were used to evaluate subjective chronotype. DNA samples were genotyped with respect to the 4-repeat and 5-repeat alleles of the VNTR PER3 polymorphism, and the genotype distribution was 192 (4-4), 191 (4-5), and 49 (5-5). The authors estimated that the power to detect an association of the 4-allele with preference for morningness or eveningness was 75%. The authors found no association between the PER3 clock gene and chronotype, indicating that the proposed role of PER3 needs further clarification.     

A variable-number taandem repeat polymorphism in PER3 is not associated with chronotype in a population with self-reported sleep problems

We examined whether a variable-number tandem repeat (VNTR) polymorphism in the circadian clock gene PER3 was associated with subjective ratings of sleep and diurnal preference in a Romanian population with high levels of self-reported sleep problems. Individuals, self-reporting to their GP for sleep disturbances, completed a battery of validated scales that assess the presence of insomnia, sleep quality and diurnal preference and had their PER3 VNTR genotype determined. We found no significant differences in chronotype, sleep quality or other psychometric measures according to PER3 VNTR and conclude that diurnal preference or self-reported sleep measures are not associated with PER3 genotype in this population.

     

Sleep,Diurnal Preference,Health, and Psychological Well-being: A Prospective Single-Allelic-Variation Study

Individual differences in sleep and diurnal preference associate with physical and mental health characteristics, but few genetic determinants of these differences have been identified. A variable number tandem repeat (VNTR) polymorphism in the PERIOD3 (PER3) gene (rs57875989) has been reported to associate with diurnal preference, i.e., preferred timing of waking and sleep. Here, the authors investigate in a prospective single-candidate genetic variant study whether allelic variation for this polymorphism associates also with reported actual sleep timing and sleep duration, as well as psychological and health measures. Six hundred and seventy-five subjects, aged 20 to 35 yrs, completed questionnaires to assess sleep and psychological and health characteristics and were genotyped for the PER3 VNTR. Homozygosity for the longer allele (PER3^5/5) of the VNTR was associated with increased morning preference, earlier wake time and bedtime, and reduced daytime sleepiness. Separate analyses of work and rest days demonstrated that the increase in time in bed during rest days was greatest in PER3^5/5 homozygotes. PER3 genotype modified the effects of sleep timing and duration on fluid intelligence and body mass index. Genotype was not associated with physical or psychological characteristics as assessed by the SF-36 Health Questionnaire, the General Health Questionnaire, the Big Five Inventory, the Behavioral Inhibition System–Behavioral Activation System scales, and the Positive and Negative Affect Scale, even though these measures varied significantly with diurnal preference as assessed by the Morningness-Eveningness Questionnaire. Whereas diurnal preference also predicts mental health and psychological characteristics, as well as sleep timing, the PER3 VNTR specifically affects measures of sleep timing and may also modify the effects of sleep on health outcome measures. (Author correspondence: a.lazar@surrey.ac.uk)     

Higher scores in the extraversion personality trait are associated with a functional polymorphism in the PER3 gene in healthy subjects

A polymorphism in the PER3 (period circadian clock 3) gene has been associated with neuropsychiatric disorders and endophenotypes. We evaluated the possible association of personality domains with the PER3 polymorphism in a sample of healthy subjects: 271 individuals were evaluated with the Big Five Inventory and genotyped for the PER3 Variable Number Tandem Repeat (VNTR) polymorphism. We found a significant association between the PER3 polymorphism and the extraversion personality trait (p = 0.0093). The 5/5 genotype carriers showed higher scores for extraversion. This is the first time that a significant association between the PER3 VNTR polymorphism and extraversion is reported.     

Individual Traits and Environmental Factors Influencing Sleep Timing: A Study of 225 Japanese Couples

Behavioral and physiological processes, such as sleep-wakefulness, thermoregulation, and hormone secretion, exhibit 24-h rhythms in most organisms. These biological rhythms are driven by the circadian clock system and are entrained by the external environment, which in the case of humans includes social time schedules. Couples might be ideal experimental subjects to discriminate between individual traits and environmental factors, as they share lifestyle habits but not genetic backgrounds. In this study, sleep timing was compared between married Japanese couples (n?=?225) who had lived together for 1 yr or more (mean 17 yrs). Additionally, the authors evaluated the influence of individual traits and environmental factors on an individual's sleep timing per each couple. The results reveal that the sleep timings of a couple are mainly associated with the chronotypes of the husband and wife, whereas the sleep timings are significantly influenced by certain environmental factors. The findings suggest that chronotype remains one of the major determinants of an individual's sleep onset and wake times. Understanding an individual's chronotype may help improve the quality of life issues surrounding sleep. (Author correspondence: mishima@ncnp.go.jp)     

PER3 Polymorphism Predicts Cumulative Sleep Homeostatic but Not Neurobehavioral Changes to Chronic Partial Sleep Deprivation

Background

The variable number tandem repeat (VNTR) polymorphism 5-repeat allele of the circadian gene PERIOD3 (PER3^5/5) has been associated with cognitive decline at a specific circadian phase in response to a night of total sleep deprivation (TSD), relative to the 4-repeat allele (PER3^4/4). PER3^5/5 has also been related to higher sleep homeostasis, which is thought to underlie this cognitive vulnerability. To date, no study has used a candidate gene approach to investigate the response to chronic partial sleep deprivation (PSD), a condition distinct from TSD and one commonly experienced by millions of people on a daily and persistent basis. We evaluated whether the PER3 VNTR polymorphism contributed to cumulative neurobehavioral deficits and sleep homeostatic responses during PSD.

Methodology/Principal Findings

PER3^5/5 (n = 14), PER3^4/5 (n = 63) and PER3^4/4 (n = 52) healthy adults (aged 22–45 y) demonstrated large, but equivalent cumulative decreases in cognitive performance and physiological alertness, and cumulative increases in sleepiness across 5 nights of sleep restricted to 4 h per night. Such effects were accompanied by increasing daily inter-subject variability in all groups. The PER3 genotypes did not differ significantly at baseline in habitual sleep, physiological sleep structure, circadian phase, physiological sleepiness, cognitive performance, or subjective sleepiness, although during PSD, PER3^5/5 subjects had slightly but reliably elevated sleep homeostatic pressure as measured physiologically by EEG slow-wave energy in non-rapid eye movement sleep compared with PER3^4/4 subjects. PER3 genotypic and allelic frequencies did not differ significantly between Caucasians and African Americans.

Conclusions/Significance

The PER3 VNTR polymorphism was not associated with individual differences in neurobehavioral responses to PSD, although it was related to one marker of sleep homoeostatic response during PSD. The comparability of PER3 genotypes at baseline and their equivalent inter-individual vulnerability to sleep restriction indicate that PER3 does not contribute to the neurobehavioral effects of chronic sleep loss.     

Period3 VNTR polymorphism influences the time-of-day pain onset of acute myocardial infarction with ST elevation

It is well established that the incidence and infarct size in acute myocardial infarction (AMI) is subject to circadian variations. At the molecular level, circadian clocks in distinct cells, including cardiomyocytes, generate 24-h cycles of biochemical processes. Possible imbalance or impairment in the cell clock mechanism may alter the cardiac metabolism and function and increase the susceptibility of cardiovascular diseases. One of the key components of the human clock system PERIOD3 (PER3) has been recently demonstrated to affect circadian expression of various genes in different tissues, including the heart. The variable number tandem repeat (VNTR) polymorphism (rs57875989) in gene Period3 (Per3) is related to multiple phenotypic parameters, including diurnal preference, sleep homeostasis, infection and cancer. The aim of our study was to investigate the effect of this polymorphism in AMI with ST elevation (STEMI). The study subjects (314 patients of Caucasian origin with STEMI, and 332 healthy controls) were genotyped for Per3 VNTR polymorphism using an allele-specific polymerase chain reaction. A gender difference in circadian rhythmicity of pain onset was observed with significant circadian pattern in men. Furthermore, the Per3^5/5 variant carriers were associated with higher levels of interleukin-6, B-type natriuretic peptide and lower vitamin A levels. By using cosinor analysis we observed different circadian distribution patterns of AMI onset at the level of genotype and allelic frequencies. Genotypes with at least one 4-repeat allele (Per3^4/5 and Per3^4/4) (N?=?264) showed remarkable circadian activity in comparison with Per3^5/5 (N?=?50), especially in men. No significant differences in genotype and/or allele frequencies of Per3 VNTR polymorphism were observed when comparing STEMI cases and controls. Our results indicate that the Per3 VNTR may contribute to modulation of cardiac functions and interindividual differences in development and progression of myocardial infarction.     

Chronotype and PERIOD3 Variable Number Tandem Repeat Polymorphism in Individual Sports Athletes

A link between diurnal preference and a variable number tandem-repeat (VNTR) polymorphism in the PERIOD3 gene (PER3) has been demonstrated: the longer PER3⁵ and shorter PER3⁴ alleles with preferences for mornings and evenings, respectively. As many competitive events in South Africa for individual athletes are scheduled for the early mornings, we hypothesized that this might favor those athletes with a preference for morning activities. Self-selected white, male cyclists (CYC, n = 125), runners (RUN, n = 120) and Ironman triathletes (IM, n = 287) of European descent were compared with a control population of active, non-competitive individuals (CON, n = 96). The chronotypes of all CYC, RUN and CON participants and a sub-sample of the IM group (n = 49) were assessed using the Horne–Östberg Morningness–Eveningness Questionnaire, and the PER3 VNTR genotype for each participant was determined. The athlete groups contained more morning-type individuals than the CON group (CYC: 72%, n = 90; RUN: 67%, n = 80; IM: 59%, n = 29; CON: 41%, n = 39; p < .001). The prevalence of the PER3⁵ allele was greater in the athlete groups (CYC: 61%, n = 152; RUN: 58%, n = 132; IM: 56%, n = 324; CON: 38%, n = 76; p < .001), and more athletes were genotyped as PER3^5/5 than CON individuals (CYC: 41%, n = 51; RUN: 23%, n = 26; IM: 28%, n = 81, CON: 9%, n = 8; p < .001). A strong relationship between chronotype and PER3 VNTR genotype was observed (p < .001). Finally, the time of day at which the athletes preferred to train was related to their chronotype (p < .001). This is the first study of its kind in a South African sporting population, and the results have not yet been replicated. These data suggest that white males of European descent participating in individual endurance sports in South Africa are more likely to be morning types. Furthermore, the PER3 VNTR may be one of the factors contributing to this observation. (Author correspondence: laura.roden@uct.ac.za)     

Phenotypic effects of genetic variability in human clock genes on circadian and sleep parameters

Circadian rhythms and sleep are two separate but intimately related processes. Circadian rhythms are generated through the precisely controlled, cyclic expression of a number of genes designated clock genes. Genetic variability in these genes has been associated with a number of phenotypic differences in circadian as well as sleep parameters, both in mouse models and in humans. Diurnal preferences as determined by the selfreported Horne-Östberg (HÖ) questionnaire, has been associated with polymorphisms in the human genes CLOCK, PER1, PER2 and PER3. Circadian rhythm-related sleep disorders have also been associated with mutations and polymorphisms in clock genes, with the advanced type cosegrating in an autosomal dominant inheritance pattern with mutations in the genes PER2 and CSNK1D, and the delayed type associating without discernible Mendelian inheritance with polymorphisms in CLOCK and PER3. Several mouse models of clock gene null alleles have been demonstrated to have affected sleep homeostasis. Recent findings have shown that the variable number tandem polymorphism in PER3, previously linked to diurnal preference, has profound effects on sleep homeostasis and cognitive performance following sleep loss, confirming the close association between the processes of circadian rhythms and sleep at the genetic level.     

Chronotype of South African adults is affected by solar entrainment

Our daily lives are influenced by three different daily timers: the solar clock, our endogenous circadian clock and the societal clock. The way an individual’s endogenous clock synchronises to the solar clock, through either advances or delays relative to sunrise and sunset, results in a phenomenon known as diurnal preference or chronotype. South Africa uses just one time zone, but in the most easterly regions of the country, the sun rises and sets up to an hour earlier than in the most westerly regions throughout the year. It was hypothesised first that South Africans living in the east of the country may have a greater preference for mornings (more morning chronotypes) than those living in the west; and second, that this difference would not be due to genetic differences in the populations, particularly a genetic polymorphism previously shown to influence chronotype. Here, we describe and compare the distribution of chorotype and PERIOD3 variable number tandem repeat (VNTR) polymorphism frequency in eastern (n = 129) and western (n = 175) sample populations. Using the Horne–Östberg Morningness, Eveningness Questionnaire we found that there was a significantly higher proportion of morning-types in the eastern population (56.6%) than in the western population (39.4%), and there were higher proportions of neither-types and evening-types in the western population (51.4% and 9.1%, respectively) than in the eastern population (37.2% and 6.2%, respectively) (p = 0.009). There were no significant differences in distribution of the PER3 genotype (p = 0.895) and allele (p = 0.636) frequencies. Although previous studies have shown associations between chronotype and PER3 VNTR genotypes, no significant associations were observed in either the eastern (p = 0.695) or the western (p = 0.630) populations. These findings indicate that, in South African populations, longitude influences chronotype independently of PER3 genotype. The impacts of the differences in chronotype whilst maintaining the same societal temporal organisation in the eastern and western regions were not assessed.     

Correlation among clock gene expression rhythms,sleep quality,and meal conditions in delayed sleep-wake phase disorder and night eating syndrome

Clock genes that comprise the circadian clock system control various physiological functions. Delayed sleep-wake phase disorder (DSWPD) and night eating syndrome (NES) are characterized by delayed sleep and meal timing, respectively. We estimated that clock gene expression rhythms in DSWPD patients may be delayed in comparison with the healthy subjects due to delayed melatonin secretion rhythms, producing eveningness chronotype in these individuals. However, it was difficult to estimate which clock gene expression rhythms were delayed or not in NES patients, because previous studies revealed that melatonin secretion rhythm was a little delayed compared with healthy individuals and that chronotype of NES patients depended on the individuals. Therefore, we examined expression rhythms of clock genes such as Period3 (Per3), nuclear receptor subfamily 1, group D, member 1 (Nr1d1) and Nr1d2 in these patients. Further, we expected sleep and meal patterns in DSWPD and NES patients may be more diverse than patterns observed in healthy subjects, and thus analyzed relationships among clock gene expression rhythms, sleep quality, sleep midpoint time, and meal times. We enrolled healthy male participants along with DSWPD and NES male patients, and asked all participants to answer questionnaires and to keep diaries to record information on their sleep and meals. Further, we asked them to collect 5–10 beard follicle samples, 6 times every 4 h. We measured clock gene expression rhythms using total RNA extracted from beard follicle cells. Peak time of clock gene expression in the NES group showed more diversity than the other groups, and that in the DSWPD group was delayed compared with the control group. In addition, the peak time of clock gene expression was negatively correlated with sleep quality and positively correlated with meal time after a long fast. Amplitudes of clock gene expression, especially Per3, positively responded to better mental and physical conditions as well as with better sleep quality. Results of this study suggest that peak times of clock gene expression in NES patients depended on the individuals; some patients with NES showed similar clock gene expression rhythm to healthy subjects, and other patients with NES showed similar to DSWPD patients. Moreover, this study suggests that meal time after a long fast may influence more determination in clock gene expression rhythms than the time of breakfast. Therefore, this study also indicates that Per3 clock gene may be one of the parameters that will help us understand sleep and meal rhythm disturbances.     

Evolutionary History of the PER3 Variable Number of Tandem Repeats (VNTR): Idiosyncratic Aspect of Primate Molecular Circadian Clock

The PER3 gene is one of the clock genes, which function in the core mammalian molecular circadian system. A variable number of tandem repeats (VNTR) locus in the 18th exon of this gene has been strongly associated to circadian rhythm phenotypes and sleep organization in humans, but it has not been identified in other mammals except primates. To better understand the evolution and the placement of the PER3 VNTR in a phylogenetical context, the present study enlarges the investigation about the presence and the structure of this variable region in a large sample of primate species and other mammals. The analysis of the results has revealed that the PER3 VNTR occurs exclusively in simiiforme primates and that the number of copies of the primitive unit ranges from 2 to 11 across different primate species. Two transposable elements surrounding the 18th exon of PER3 were found in primates with published genome sequences, including the tarsiiforme Tarsius syrichta, which lacks the VNTR. These results suggest that this VNTR may have evolved in a common ancestor of the simiiforme branch and that the evolutionary copy number differentiation of this VNTR may be associated with primate simiiformes sleep and circadian phenotype patterns.     

Characterization of bimodal chronotype and its association with sleep: A population-based study

The circadian system coordinates internal events in a daily schedule to make sure that the body systems are synchronized to environmental time and internal cues. One important behavioral aspect of the circadian system is the chronotype. It is usually assessed through subjective questionnaires, being the Horne-Ostberg Morningness–Eveningness Questionnaire (MEQ) one of the most used. It classifies individuals into three major categories: morning, evening, and intermediate types. Recently, it has been hypothesized the existence of a fourth chronotype, the bimodal type, through an algorithm derived from the MEQ responses. Bimodals answer as morning-types in some questions, and as evening-types in others, resulting in an intermediate total score. To better characterize this phenotype, the present study aimed to detect and characterize the frequency of the bimodal chronotype in the EPISONO, a large population-based cohort, as well as to verify the association between bimodality and sleep parameters and genetic variation in the PER3 gene. Of the 1,042 individuals who participated of the EPISONO, 857 had MEQ filled correctly. We found that 16% of our sample were bimodal types. We observed that bimodal individuals were significantly younger and had lower body mass index. The association between PER3 VNTR genotype and gender with bimodal chronotype was not significant. However, we found an association between bimodality and Epworth Sleepiness Scale (EES) and apnea-hypopnea index (AHI). We did not find a statistically significant difference between bimodals and intermediate non-bimodals for the studied variables. Lastly, it was observed that the most significant predictors for bimodal chronotype were female gender, AHI, and EES. In conclusion, the present work provides more evidence that the bimodal type might have to be considered when classifying chronotype and its association with young age and sleepiness may be due to the influence of social and environmental factors.     

Exploring the role of circadian clock gene and association with cancer pathophysiology

ABSTRACT

Most of the processes that occur in the mind and body follow natural rhythms. Those with a cycle length of about one day are called circadian rhythms. These rhythms are driven by a system of self-sustained clocks and are entrained by environmental cues such as light-dark cycles as well as food intake. In mammals, the circadian clock system is hierarchically organized such that the master clock in the suprachiasmatic nuclei of the hypothalamus integrates environmental information and synchronizes the phase of oscillators in peripheral tissues.

The circadian system is responsible for regulating a variety of physiological and behavioral processes, including feeding behavior and energy metabolism. Studies revealed that the circadian clock system consists primarily of a set of clock genes. Several genes control the biological clock, including BMAL1, CLOCK (positive regulators), CRY1, CRY2, PER1, PER2, and PER3 (negative regulators) as indicators of the peripheral clock.

Circadian has increasingly become an important area of medical research, with hundreds of studies pointing to the body’s internal clocks as a factor in both health and disease. Thousands of biochemical processes from sleep and wakefulness to DNA repair are scheduled and dictated by these internal clocks. Cancer is an example of health problems where chronotherapy can be used to improve outcomes and deliver a higher quality of care to patients.

In this article, we will discuss knowledge about molecular mechanisms of the circadian clock and the role of clocks in physiology and pathophysiology of concerns.     

Effect of sleep deprivation on rhythms of clock gene expression and melatonin in humans

This study investigated the impact of sleep deprivation on the human circadian system. Plasma melatonin and cortisol levels and leukocyte expression levels of 12 genes were examined over 48?h (sleep vs. no-sleep nights) in 12 young males (mean?±?SD: 23?±?5 yrs). During one night of total sleep deprivation, BMAL1 expression was suppressed, the heat shock gene HSPA1B expression was induced, and the amplitude of the melatonin rhythm increased, whereas other high-amplitude clock gene rhythms (e.g., PER1-3, REV-ERBα) remained unaffected. These data suggest that the core clock mechanism in peripheral oscillators is compromised during acute sleep deprivation.     

ASSOCIATIONS OF METABOLIC PARAMETERS AND ETHANOL CONSUMPTION WITH MESSENGER RNA EXPRESSION OF CLOCK GENES IN HEALTHY MEN

Recent studies suggest that the impairment of circadian clock function causes various pathological conditions, such as obesity, diabetes, and alcoholism, and an altered mRNA expression of clock genes was found under these conditions. However, it remains to be determined whether clock gene expression varies depending on metabolic conditions even in healthy people. To address this issue, we investigated the associations of metabolic parameters and alcohol consumption with mRNA expression of clock genes (CLOCK, BMAL1, PER1, PER2, and PER3) in peripheral blood cells obtained from 29 healthy non-obese elderly men (age 51–78 yrs) who adhered to a regular sleep-wake routine, through a single time-of-day venous blood sampling at ～09:00?h. There were significant correlations between (1) waist circumference and mRNA level of PER1 (r?=?0.43), (2) plasma glucose concentration and PER2 (r?=?0.50), (3) ethanol consumption and BMAL1 (r?=?0.43), and (4) serum γ-GTP concentration (a sensitive marker of alcohol consumption) and PER2 (r?=?0.40). These results suggest mRNA expression of clock genes is associated with obesity, glucose tolerance, and ethanol consumption even in healthy people. (Author correspondence: akiofuji@jichi.ac.jp)     

Sleep quality and methylation status of core circadian rhythm genes among nurses and midwives

Poor sleep quality or sleep restriction is associated with sleepiness and concentration problems. Moreover, chronic sleep restriction may affect metabolism, hormone secretion patterns and inflammatory responses. Limited recent reports suggest a potential link between sleep deprivation and epigenetic effects such as changes in DNA methylation profiles. The aim of the present study was to assess the potential association between poor sleep quality or sleep duration and the levels of 5-methylcytosine in the promoter regions of PER1, PER2, PER3, BMAL1, CLOCK, CRY1 CRY2 and NPAS2 genes, taking into account rotating night work and chronotype as potential confounders or modifiers. A cross-sectional study was conducted on 710 nurses and midwives (347 working on rotating nights and 363 working only during the day) aged 40–60 years. Data from in-person interviews about sleep quality, chronotype and potential confounders were used. Sleep quality and chronotype were assessed using Pittsburgh Sleep Quality Questionnaire (PSQI) and Morningness–Eveningness Questionnaire (MEQ), respectively. Morning blood samples were collected. The methylation status of the circadian rhythm genes was determined via quantitative methylation-specific real-time PCR assays (qMSP) reactions using DNA samples derived from leucocytes. The proportional odds regression model was fitted to quantify the relationship between methylation index (MI) as the dependent variable and sleep quality or sleep duration as the explanatory variable. Analyses were carried out for the total population as well as for subgroups of women stratified by the current system of work (rotating night shift/day work) and chronotype (morning type/intermediate type/evening type). A potential modifying effect of the system of work or the chronotype was examined using the likelihood ratio test. No significant findings were observed in the total study population. Subgroup analyses revealed two statistically significant associations between a shorter sleep duration and 1) methylation level in PER2 among day workers, especially those with the morning chronotype (OR = 2.31, 95%CI:1.24–4.33), and 2) methylation level in CRY2 among subjects with the intermediate chronotype, particularly among day workers (OR = 0.52, 95%CI:0.28–0.96). The study results demonstrated a positive association between average sleep duration of less than 6 hours and the methylation level of PER2 among morning chronotype subjects, and an inverse association for CRY2 among intermediate chronotype subjects, but only among day workers. Both the system of work and the chronotype turned out to be important confounders and modifiers in a number of analyses, making it necessary to consider them as potential covariates in future research on sleep deficiency outcomes. Further studies are warranted to explore this under-investigated topic.