Rhythmic expression of circadian clock genes in human leukocytes and beard hair follicle cells.

Evaluating individual circadian rhythm traits is crucial for understanding the human biological clock system. The present study reports characterization of physiological and molecular parameters in 13 healthy male subjects under a constant routine condition, where interfering factors were kept to minimum. We measured hormonal secretion levels and examined temporal expression profiles of circadian clock genes in peripheral leukocytes and beard hair follicle cells. All 13 subjects had prominent daily rhythms in melatonin and cortisol secretion. Significant circadian rhythmicity was found for PER1 in 9 subjects, PER2 in 3 subjects, PER3 in all 13 subjects, and BMAL1 in 8 subjects in leukocytes. Additionally, significant circadian rhythmicity was found for PER1 in 5 of 8 subjects tested, PER2 in 2 subjects, PER3 in 6 subjects, and BMAL1 in 3 subjects in beard hair follicle cells. The phase of PER1 and PER3 rhythms in leukocytes correlated significantly with that of physiological rhythms. Our results demonstrate that leukocytes and beard hair follicle cells possess an endogenous circadian clock and suggest that PER1 and PER3 expression would be appropriate biomarkers and hair follicle cells could be a useful tissue source for the evaluation of biological clock traits in individuals.

Validity of the Japanese version of the Munich ChronoType Questionnaire.

To assess circadian preference with a score, the Morningness-Eveningness Questionnaire (MEQ) has been used for more than 3 decades now. More recently, the Munich ChronoType Questionnaire (MCTQ) was developed: it asks for sleep-wake behavior on work and free days and uses the midpoint of sleep on free days (MSF), corrected for sleep debt accumulated during the work week as an indicator of chronotype (MSFsc). In this study, we developed a Japanese version of the MCTQ by using a translation/back-translation approach including an examination of its semantic validity. In a subsequent questionnaire survey, 450 adult men and women completed the Japanese versions of the MCTQ and MEQ. Results showed that MEQ scores were significantly negatively correlated with mid-sleep parameters assessed by the MCTQ, on both, work and free days, as well as with the chronotype measure MSFsc (r = -0.580 to -0.652, all p < 0.001). As in the original German version, the strongest correlation was observed between MEQ score and MSF. A physiological validation study using dim light melatonin onset as a circadian phase marker (N = 37) showed a high correlation between chronotype as assessed with the MSFsc (r = 0.542, p < 0.001), and less so for MEQ score (r = -0.402, p = 0.055). These results demonstrate the validity of the Japanese MCTQ and provide further support of the adequacy of the MCTQ as a chronotype measure.

Screening of clock gene polymorphisms demonstrates association of a PER3 polymorphism with morningness-eveningness preference and circadian rhythm sleep disorder.

A system of self-sustained biological clocks controls the 24-h rhythms of behavioral and physiological processes such as the sleep-wake cycle. The circadian clock system is regulated by transcriptional and translational negative feedback loops of multiple clock genes. Polymorphisms in circadian clock genes have been associated with morningness-eveningness (diurnal) preference, familial advanced sleep phase type (ASPT), and delayed sleep phase type (DSPT). We genotyped single-nucleotide polymorphisms in circadian clock genes in 182 DSPT individuals, 67 free-running type (FRT) individuals, and 925 controls. The clock gene polymorphisms were tested for associations with diurnal preference and circadian rhythm sleep disorder (CRSD) phenotypes. The PER3 polymorphism (rs228697) was significantly associated with diurnal preference and the FRT phenotype. The minor allele of rs228697 was more prevalent in evening types than in morning types (sex-adjusted odds ratio (OR), 2.483, Bonferroni-corrected P = 0.012) and in FRT individuals compared with the controls (age- and sex-adjusted OR, 2.021, permutated P = 0.017). Our findings support the notion that PER3 polymorphisms could be a potential genetic marker for an individual's circadian and sleep phenotypes.

In vitro circadian period is associated with circadian/sleep preference.

Evaluation of circadian phenotypes is crucial for understanding the pathophysiology of diseases associated with disturbed biological rhythms such as circadian rhythm sleep disorders (CRSDs). We measured clock gene expression in fibroblasts from individual subjects and observed circadian rhythms in the cells (in vitro rhythms). Period length of the in vitro rhythm (in vitro period) was compared with the intrinsic circadian period, τ, measured under a forced desynchrony protocol (in vivo period) and circadian/sleep parameters evaluated by questionnaires, sleep log, and actigraphy. Although no significant correlation was observed between the in vitro and in vivo periods, the in vitro period was correlated with chronotype, habitual sleep time, and preferred sleep time. Our data demonstrate that the in vitro period is significantly correlated with circadian/sleep preference. The findings suggest that fibroblasts from individual patients can be utilized for in vitro screening of therapeutic agents to provide personalized therapeutic regimens for CRSD patients.