Figure 1
Sample collection schedule of healthy participants.
Participants arrived at the CTRC 9:00–9:30 on scheduled date and stayed in the room until about 6:00 the next day for sample collection. From each participant, cheek cells were collected every four hours (green) between about 10:00 on day 1 and 6:00 on day 2; saliva was collected every hour, starting from about 6 hours before their usual bedtime (10 p.m. in this example) until 2 hours after their usual bedtime, under dim light (light exposure lower than 5 lux). Blood was collected about 10:00.
Table 1
Table 1 Demographic information of study participants.
| VARIABLES | NO. | % | MEAN (STD), MIN, MAX |
|---|---|---|---|
| Age (years) | |||
| 23–30 | 4 | 36.4 | |
| 31–50 | 4 | 36.4 | |
| 51–60 | 3 | 27.3 | |
| Sex | |||
| Women | 8 | 72.7 | |
| Men | 3 | 27.3 | |
| Race | |||
| Asian | 2 | 18.2 | |
| Black | 3 | 27.3 | |
| Hispanic | 1 | 9.1 | |
| White | 5 | 45.5 | |
| Chronotype defined by the morning-evening questionnaire scores (Horne and Ostberg, 1976, ref 23) | |||
| Moderate evening: 31–41 | 1 | 9.1 | |
| Intermediate: 42–58 | 4 | 36.4 | |
| Moderate morning: 59–69 | 5 | 45.5 | |
| Definite morning: 70–86 | 1 | 9.1 | |
| DLMO (3pg/ml) time (HH;MM) | 20:13 (1:25), 17:22, 22:04 | ||
| Bedtime-PSQI* (HH:MM) | 22:42 (1:18), 20:30, 25:30 | ||
| Waketime-PSQI* (HH:MM) | 6:24 (0:36), 5:45, 7:45 | ||
| Midsleep-PSQI* (HH:MM) | 2:24 (0:42), 1:15, 3:15 | ||
| 5-day average weekday midsleep – Actigraphy** (HH:MM) | 3:01 (0:48), 1:56, 4:07 | ||
| 2-day average weekend midsleep – Actigraphy** (HH:MM) | 3:39 (1:46), –0:28, 6:01 | ||
| 5-day average weekday waketime – Actigraphy** (HH:MM) | 6:25 (0:47), 5:31, 7:57 | ||
| 2-day average weekend waketime-Actigraphy** (HH:MM) | 7:16 (1:17), 3:19, 8:40 | ||
| Intervals between completion of actigraphy data collection and sample collection | |||
| <7 days | 5 | 45.5 | |
| 11 | 1 | 9.1 | |
| 15–19 | 3 | 27.3 | |
| >21 | 2 | 18.2 | |
[i] DLMO: Dim Light Melatonin Onset based on thresholds of 3pg/ml.
* Sleep times from the Pittsburg Sleep Quality Index Questionnaire responses.
** Sleep times based on the actigraphy measurements over seven days.
Table 2
Genes in the circadian and cell cycle pathways with p-value ≤ 0.1 and amplitude > 0.1.
| GENE | AMP | RAMP | CLOCK PEAK TIME* | PEAK TIME RANGE | P-VALUE | |||||
|---|---|---|---|---|---|---|---|---|---|---|
| POINT ESTIMATE | AVERAGE | 95%CI | TIME IN CLOCK | DLMO-ADJ** | MIDSLEEP-ADJ** | |||||
| CIART | 0.36 | 0.66 | 10.4 | 10.3 | 8.7–11.8 | 6.7–14.5 (7.8) | 10.7 | 9.3 | 0.008† | |
| WEE1 | 0.18 | 0.21 | 12.5 | 12.9 | 9.6–16.2 | 2.1–18.8 (16.7) | 15.1 | 15.6 | 0.009 | |
| PER3 | 0.27 | 0.50 | 10.0 | 10.1 | 8.6–11.6 | 6.3–14.4 (8.1) | 8.3 | 8.6 | 0.009† | |
| PER1 | 0.3 | 0.22 | 10.8 | 10.6 | 8.8–12.4 | 6.2–14.2 (8.0) | 10.3 | 9.2 | 0.08 | |
| ARNTL | 0.34 | 1.3 | 19.5 | 19.4 | 15.8–22.6 | 13.8–30.0 (16.2) | 18.7 | 17.8 | 0.08 | |
| TEF | 0.44 | –1.76 | 10.3 | 11.4 | 9.0–13.7 | 6.7–18.1 (11.4) | 0.09 | |||
| CRY2 | 0.25 | 0.32 | 13.5 | 13.0 | 11.0–14.9 | 8.0–17.5 (8.5) | 0.09 | |||
| PER2 | 0.31 | 0.51 | 13.1 | 12.5 | 10.5–14.6 | 6.5–18.0 (11.5) | 0.1 | |||
[i] AMP: the amplitude is the half difference between the highest and lowest values of the log10(TPM) gene expression levels, based on the fitted MetaCycle harmonic model.
rAMP (the relative amplitude): the ratio between the amplitude and baseline values (when the baseline absolute value is greater than 1).
* Peak time point-estimate was based on the aggregated peak-time calculated using meta3d output; Average and 95%CI was based on individual participant peak-time values.
** Peak times of gene expression adjusted by DLMO or Midsleep for each participant was calculated by the gene peak time in clock minus each person’s DLMO time or PSQI-midsleep time.
† False Discovery Rate < 0.1 adjusting for number genes in the circadian rhythm pathway.
Figure 2
mRNA expression levels (LogTPM) over 20 hours for selected genes with most significant circadian variation. Numbers are Participant ID.
Figure 3
Estimated peak times and amplitudes of selected genes for each participant (n = 11). Peak times are indicated by the clock times; amplitudes are indicated by the distance from the center. The numbers are the last two digits of the Participant IDs listed in Figure 2.
Figure 4
Heatmap of mRNA gene expression among genes in the circadian rhythm pathway and WEE1. Correlation coefficients are indicated using both numbers and colors.
Figure 5
Correlations between peak times of selected circadian genes, with sleep times and the DLMO time.
Table 3
Partial Spearman correlation between gene peak times and age, and sleep time*.
| PER1 | PER2 | PER3 | CIART | ||
|---|---|---|---|---|---|
| age | Coefficient | 0.77 | –0.50 | 0.69 | 0.25 |
| p value | 0.03 | 0.21 | 0.06 | 0.55 | |
| PSQI_midsleep** | Coefficient | 0.23 | –0.27 | –0.43 | –0.48 |
| p value | 0.58 | 0.52 | 0.29 | 0.23 |
[i] * Analyses adjusted for age, sex, sleep time, and DLMO, excluding the variable that was being examined.
** mid-time between the bedtime and wake-up time from the Pittsburg Sleep Quality Index Questionnaire.