Molecular-timetable methods for detection of body time and rhythm disorders from single-time-point genome-wide expression profiles

Abstract

Detection of individual body time (BT) via a single-time-point assay has been a longstanding unfulfilled dream in medicine, because BT information can be exploited to maximize potency and minimize toxicity during drug administration and thus will enable highly optimized medication. To achieve this dream, we created a ''molecular timetable'' composed of >100 ''time-indicating genes,'' whose gene expression levels can represent internal BT. Here we describe a robust method called the ''molecular-timetable method'' for BT detection from a single-time-point expression profile. The power of this method is demonstrated by the sensitive and accurate detection of BT and the sensitive diagnosis of rhythm disorders. These results demonstrate the feasibility of BT detection based on single-time-point sampling, suggest the potential for expression-based diagnosis of rhythm disorders, and may translate functional genomics into chronotherapy and personalized medicine.DNA microarray ͉ gene expression ͉ chronotherapy D iverse physiological and metabolic processes exhibit circadian rhythms, which are endogenous self-sustained oscillations with a period of Ϸ24 hours. In mammals, several clock genes, including Clock, Bmal1͞Mop3, Per1, Per2, Cry1, Cry2, Cki, and RevErbA␣, including Dbp, E4bp4, Dec1͞Stra13, Dec2, Per3, Npas2, RevErbA␤, Ror␣, Ror␤, and Ror␥, regulate, at least in part, gene expression in central and͞or peripheral clocks (1). Reflecting the temporal changes in gene expression in central and peripheral clocks (2-5), the potency and͞or toxicity in drug administration depend on an individual's body time (BT) (6-10). It has been suggested that drug administration at the appropriate BT can improve the outcome of pharmacotherapy by maximizing potency and minimizing the toxicity of the drug (11), whereas drug administration at an inappropriate BT can induce severe side effects (12). Despite the effectiveness and importance of such BT-dependent therapy, termed ''chronotherapy'' (6-10), its clinical use has been obstructed by the lack of clinically applicable methods for BT detection. To address these difficulties, we attempted to create standard expression profiles, termed a ''molecular timetable,'' composed of Ͼ100 ''time-indicating genes'' and their expression levels during the course of a day, and then to apply this timetable to BT detection. Materials and MethodsAnimals. To select time-indicating genes and construct their standard expression profiles, we analyzed the previously obtained genome-wide expression profiles (5) from pooled livers of four Balb͞c mice (male) every 4 h over 2 d under 12-h light͞12-h dark (LD) or constant-dark (DD) conditions. We independently sampled livers from eight individual Balb͞c mice (male) at Zeitgeber time (ZT)12 (n ϭ 4), ZT6 (n ϭ 1), ZT18 (n ϭ 1), Circadian time (CT)6 (n ϭ 1), and CT18 (n ϭ 1) to verify the capability of the molecular-timetable method. ZT is used for a timescale under LD conditions, whereas CT is used for a timescale under DD conditions. ZT0 represents lights on, and ...

Human blood metabolite timetable indicates internal body time

“…Identification of the circadian-oscillating metabolites is important for connecting our current metabolite timetable of human blood to previously reported circadian biology in humans. Identification is also important for further development of our method because once a metabolite is identified, we can update the circadianmetabolite reference list in our human blood timetable, which will improve the sensitivity and specificity of the molecular timetable (12,42). Therefore, we attempted to identify several circadian-oscillating metabolites in constructing our metabolite timetable and found that a large fraction belong to the steroid hormone metabolism pathway (Fig.…”

“…We note that a molecular timetable concept can be applied not only for circadian-oscillating metabolites, but also for other circadian-oscillating substances (12,42) and in other tissues like human hair follicle cells from the head or chin (63). Comparing molecular timetable methods with different reference molecules such as clock-controlled RNAs in humans will be of interest to reveal internal synchronization (or desynchronization) between different peripheral clocks such as blood clocks and skin clocks.…”

“…Compared with the conventional method, the molecular-timetable method requires only one or few time-point samplings. In mice, this method works with the expression activity of clock-controlled genes in different organs (42,43) and with oscillatory metabolites in blood plasma (12).…”

“…Although this strategy directly measures internal body time, it requires labor-intensive constant sampling under controlled environmental conditions to reveal metabolite peaks and rhythms. To reduce this burden, we previously developed a molecular-timetable method (12,42), which was inspired by Linné's flower clock (Fig. 1A).…”

Noninvasive method for assessing the human circadian clock using hair follicle cells

“…We moreover succeeded in single-point phase prediction using expression data for nine clock genes obtained from mouse peripheral tissues; this suggests that even one-point phase prediction may be possible for human data with our standard-curve method, using more genes whose rhythms of expression are consistently detectable. There is a report on a single-point phase prediction method using expression profiles of more than 100 circadian output genes in mouse liver (23).…”