Skip to main content
NCBI home page
UKPMC Funders Author Manuscripts logoLink to UKPMC Funders Author Manuscripts
. Author manuscript; available in PMC: 2021 Oct 23.
Published in final edited form as: J Sleep Res. 2007 Mar 1;16(1):12–6. doi: 10.1111/j.1365-2869.2007.00561.x

Age-related change in the association between a polymorphism in the PER3 gene and preferred timing of sleep and waking activities

Kay H S Jones 1, Jason Ellis 1, Malcolm Von Schantz 1, Debra J Skene 1, Derk-Jan Dijk 1, Simon N Archer 1,✉
PMCID: PMC7611878  EMSID: EMS137085  PMID: 17309758

Summary

The objective of this study was to investigate the effect of age on the association between preferred timing of sleep and waking activities and a coding-region variable number tandem repeat (VNTR) polymorphism in the clock gene PER3. We have previously reported this polymorphism to associate with diurnal preference and delayed sleep phase syndrome (DSPS). Participants (n = 1590; 707 males and 883 females) completed the Horne–Östberg (HÖ) questionnaire for diurnal preference and provided a DNA sample. Overall HÖ scores were plotted against age. The 5% extremes and intermediates were selected for genotyping. Frequencies of the PER3 4- and 5-repeat alleles were examined in separate age groups (18–29, 30–39, 40–49 and 50+ years of age). The 4-repeat allele was significantly more frequent in evening types, and the 5-repeat allele more frequent in morning types (Fisher’s exact test, P = 0.016). Analysis in the four age groupings revealed that the strength of this association attenuated with age and was significant only in the youngest group (18–29 years). These results extend our previous finding of an association between the PER3 VNTR and diurnal preference. They also demonstrate that diurnal preference in young people is more closely associated with this polymorphism than it is in other age groups.

Keywords: circadian rhythms, diurnal preference, genetic, polymorphism, sleep, tandem repeat sequences

Introduction

The circadian pacemaker and sleep homeostasis are considered to be the main physiological processes that determine sleep timing. Variation in the timing of sleep and waking activities may be related to variations in key parameters of the circadian timing system, including its sensitivity to light and its endogenous period. The Horne–Östberg (HÖ) questionnaire provides a quantitative measure of diurnal preference (Horne and Östberg, 1976). The HÖ score has been validated by the demonstration that it correlates with the timing of the: (i) core body temperature rhythm, and melatonin rhythm as assessed during constant routine protocols (i.e. in the absence of the masking effects of the sleep–wake and light–dark cycle) (Duffy et al., 1999) and (ii) sleep–wake cycle (Kerkhof and Van Dongen, 1996; Taillard et al., 2003). The HÖ questionnaire consists of 19 items relating, for example, to time of day preference for exercise and mental alertness, as well as levels of tiredness upon waking and before going to bed. Internal consistency has been reported to be high (α = 0.82). Thirty years later, the original 19-item scale remains the most widely used tool for measuring diurnal preference.

Several parameters affect diurnal preference. Evidence for a genetic contribution has been derived from twin studies. Monozygotic twins are more likely to exhibit shared diurnal preference than dizygotic twins, up to 50% of the variance being estimated to be accounted for by heritability (Vink et al., 2001). Gender also affects diurnal preference, females having been shown to have a higher tendency towards morningness than males (Adan and Natale, 2002; Robilliard et al., 2002; Vink et al., 2001).

Age is another major factor relating to HÖ score. Several reports demonstrate that morningness increases with age (Carrier et al., 1997; Hur et al., 1998; Robilliard et al., 2002; Taillard et al., 2004; Tankova et al., 1994). It has been suggested that this follows an eveningness peak reached at the end of adolescence (Roenneberg et al., 2004). The age-related shift towards higher scores (i.e. morningness) on the HÖ scale is associated with earlier wake times and earlier timing of the endogenous rhythms of core body temperature and melatonin (Duffy et al., 1999). Analysis of the factors underlying these age-related changes has indicated that they are only partially related to changes in parameters of the circadian pacemaker. Age-related changes in sleep need/duration are also thought to contribute considerably to these age-related changes in diurnal preference (Dijk et al., 2000; Duffy et al., 1999). Some studies have developed specific tools to address these age-related changes (Paine et al., 2006) and to assess chronotype accurately from changing patterns of sleep/wake schedules due to social factors (Roenneberg et al., 2003).

Diurnal preference has also been investigated in relation to lifestyle regularity using the Composite Scale of Morningness and Social Rhythm Metric diary (SRM-5) (Monk et al., 2004). Morning types have been reported as having higher scores on the SRM-5, indicating greater daily lifestyle regularity than intermediate or evening types, with evening types having the lowest scores and therefore the least daily regularity (Monk et al., 2004). Eveningness has been linked to increased impulsivity (Caci et al., 2005), which suggests that diurnal preference may have implications for psychiatric evaluations and measurements.

Studies investigating clock gene polymorphisms and circadian phenotype have demonstrated that polymorphisms in the clock gene PER3 are associated with diurnal preference and may be linked with circadian rhythm disorders such as delayed sleep phase syndrome (DSPS) (Archer et al., 2003; Ebisawa et al., 2001). The coding region of the PER3 gene contains a variable number tandem repeat (VNTR) which has evolved in primates, with inter- and intra-specific length polymorphism (Jenkins et al., 2005). In humans, we have reported the longer allele (five tandem repeats) to associate with morningness, and the shorter allele (four tandem repeats) with eveningness and DSPS (Archer et al., 2003). This polymorphism does not appear to show a significant latitudinal cline or balancing selection (Nadkarni et al., 2005), but the same association with diurnal preference (although not with DSPS) has been confirmed in a Brazilian population (Pereira et al., 2005).

The aim of the present study was to explore further the association between the reported PER3 VNTR polymorphism and diurnal preference in a larger sample. The effect of age on the allele frequencies within populations with extreme morning or evening preference was also examined. The allele frequencies within populations with extreme morning or evening preference were examined in different age groups.

Methods

Participants

Visitors to the London Science Museum participated in the study either in 2001 (n = 484; 217 males and 267 females) (Robilliard et al., 2002), or in 2004 (n = 1106; 490 males and 616 females) and were aged between 18 and 81 years. In the more recent study, in analogy with the previous one, volunteers were recruited from the visitor population at the Science Museum over a 9-week period (September–November 2004) as part of the Live Science exhibition in the Wellcome Wing of the Science Museum. All visitors were invited to participate regardless of country of residence. Multiple members of the same family were excluded from the genotyping part of the study. Written informed consent was obtained from all volunteers after explanation of the project rationale. This study complied with the Declaration of Helsinki, and approval was granted by the University of Surrey Ethics Committee.

Participants completed a computer-based version of the HÖ diurnal preference questionnaire, and received a print-out of their score. They were also asked to provide a buccal swab (MasterAmp Catch-All Sample Collection Swabs; Epicentre Biotechnologies, Madison, WI, USA) for DNA extraction, and to complete a short hard copy questionnaire relating to their usual sleep habits, such as usual bedtimes, sleep latency, number of awakenings etc.

Genotyping

Horne–Östberg scores were plotted against age and a linear regression line was applied to the plot. Those participants who were outside of the 5% cut-off lines parallel to the regression line (i.e. those with the most extreme scores of morningness or eveningness, n = 80 in each group) were selected for genotyping. Participants falling on, or very close to, the regression line were selected as representative intermediate samples (n = 80). DNA was extracted from the selected groups using BuccalAmp DNA Extraction Kits (Epicentre Biotechnologies). Genotyping was performed as described previously (Archer et al., 2003). Allele frequency associations were analysed using Fisher’s exact test (two-sided, Instat version 3.01; Graphpad Software, San Diego, CA, USA).

Results

Description of the sample

Morningness, as measured by the HÖ questionnaire, increased with age (linear regression F = 198.77; y = 0.2749x + 42.193; d.f. = 1; P < 0.05). There were no significant differences found between the 2001 and 2004 data sets for age [t(1,590) = 0.95, P > 0.05] nor HÖ score [t(1,590) = 0.13, P > 0.05]. Similarly, there was no significant difference between the two data sets for gender (χ 2 = 0.85, d.f. = 1, P > 0.05). The combined HÖ frequency distribution was tested for normality using the Kolmogorov–Smirnov test and was found not to satisfy the conditions for assumed normality, exhibiting skewness towards higher HÖ score (skewness = −0.088; kurtosis = −0.78; P < 0.05). The extreme morning and evening types (n = 80 in each group) were tested for differences in the number of males and females comprising the extreme distributions. The results of the χ 2-test showed that no significant gender differences existed (χ 2 = 2.30, d.f. = 1, P > 0.05).

Examined independently, females had a mean HÖ score (±standard deviation) of 52.4 ± 11.9, whilst males had a mean score of 51.4 ± 11.0. Both distributions taken from the entire sample (n = 1590) were tested separately for normality, and were found not to conform to a normal distribution, with females showing more skewness towards higher scores (Kolmogorov–Smirnov; skewness = −0.155 for females; −0.074 for males; kurtosis = −0.458 for females; −0.514 for males; P < 0.05). The male and female distributions were analysed for differences between the genders with the Mann–Whitney test and found to be not significantly different from each other (u = 295 912; Z = −1.78; P = 0.07).

Analysis of PER3 genotype versus HÖ score

The frequencies of the 4- and 5-repeat alleles in the 5-percentile of individuals with extreme morning and evening preference were tested for associations. A significant association between PER3 VNTR genotype and diurnal preference was found (Fisher’s exact P = 0.016, odds ratio = 0.53, 95% confidence interval 0.33–0.85). The 4-repeat allele was more strongly associated with evening types (0.75 in evening types; 0.62 in morning types) and the 5-repeat allele with morning types (0.38 in morning types; 0.25 in evening types). Similarly, Fisher’s exact test was performed on the intermediate types compared to extreme morning and evening types (4-repeat = 0.63, 5-repeat = 0.37). No significant difference was found between the allele frequencies in morning types and intermediates (Fisher’s exact P = 0.91, odds ratio = 0.95, 95% confidence interval 0.60–1.49). However, a significant difference was found between intermediate and evening types (Fisher’s exact P = 0.02, odds ratio = 0.56, 95% confidence interval 0.34–0.90). The observed genotype frequencies for the extreme diurnal preference groups were found to be in Hardy–Weinberg equilibrium (Hardy, 1908). When males and females were analysed separately, the distribution of allele frequencies for each gender was consistent with those found in the combined sample. This suggests that gender did not affect the association between the PER3 polymorphism and diurnal preference.

The effect of age on the 4-repeat allele frequency was investigated in the 5% extremes (Fig. 1). The plot showed that as age increased, the frequency of the 4-repeat allele became closer to the population mean of 0.68 (Archer et al., 2003; Nadkarni et al., 2005), decreasing in evening types, and increasing in morning types. Chi-squared analysis revealed an overall significant difference in allele frequency between the extreme morning and evening types when examined across age groups (χ 2 = 13.95, d.f. = 3, P < 0.001). Further analysis revealed that there was a clear separation (deviation from the population mean) in phenotypes of younger subjects (18–29 years old). This deviation decreased with increasing age, so that in the 40 to 49-year-old group both extremes had allele frequencies that were indistinguishable from each other and from the combined population mean. In subjects aged 50 and over, an apparent separation was visible again. Fisher’s exact test was performed on these data, and a significant difference was found between allele frequencies in the morning and evening types at age 18–29 years only (odds ratio = 0.34; 95% confidence interval = 0.132–0.890; P = 0.036). Similarly, when the high versus low scores (based upon the mean allele frequency score) were compared against an age dimension of younger (18–39 years) and older (40+ years) subjects a significant difference was found between these two groups (χ 2 = 27.23, d.f. = 3, P < 0.001). No significant association was observed in the other age groups. The age, HÖ score and genotype distribution for the eight sub-groups is shown in Table 1.

Figure 1.

Figure 1

Open in a new tab

Frequency of the 4-repeat allele across age groups in extreme morning (filled circles) and evening (open circles) types. The dotted line represents the combined population mean for the 4-repeat allele frequency (0.68). Asterisk denotes a statistically significant association between allele frequency and diurnal preference.

Table 1. Age, Horne–Östberg (HÖ) score and genotype distributions across age ranges.

Morning types Evening types
Age range HÖ score range Genotype frequency HÖ score range Genotype frequency
18–29 65–78 (n = 21) 4/4 = 0.38 21–29 (n = 22) 4/4 = 0.64
4/5 = 0.38 4/5 = 0.32
5/5 = 0.24 5/5 = 0.04
30–39 67–83 (n = 25) 4/4 = 0.40 21–33 (n = 21) 4/4 = 0.57
4/5 = 0.40 4/5 = 0.33
5/5 = 0.20 5/5 = 0.10
40–49 69–80 (n = 24) 4/4 = 0.46 25–35 (n = 11) 4/4 = 0.45
4/5 = 0.46 4/5 = 0.45
5/5 = 0.08 5/5 = 0.10
50+ 70–78 (n = 10) 4/4 = 0.30 25–44 (n = 26) 4/4 = 0.58
4/5 = 0.50 4/5 = 0.34
5/5 = 0.20 5/5 = 0.08
Open in a new tab

Discussion

Data from a large sample of 1590 participants [including n = 484 from our previous study (Robilliard et al., 2002)] showed a significant association between diurnal preference and the PER3 VNTR polymorphism, with the shorter allele (4-repeat) being associated with eveningness and the longer allele (5-repeat) associating with morningness. This is in accordance with our previous report (Archer et al., 2003) and an independent replication in a Brazilian population (Pereira et al., 2005). Females appeared to be more skewed towards morningness than males, as has been reported previously (Adan and Natale, 2002; Robilliard et al., 2002), although the differences between the distributions of males and females were not statistically significant. The trend for increasing morningness with age was also observed within these data.

When different age groups of extreme morning and evening individuals were examined in relation to PER3 genotype (using allele frequency of the 4-repeat allele) an effect of age on allele frequency was found. There was a significant overall difference between the extreme morning and evening types in terms of their allele frequency across age. More specifically, younger subjects (18–29 years) with extreme diurnal preference displayed a significant deviation from the population mean, indicating that their diurnal preference may be more strongly influenced by their genotype and less influenced by exogenous factors. With increasing age, the allele frequency became closer to the mean population frequency of 0.68. This finding suggests that the diurnal preference of older people, particularly within the 40–49-year age range, may not be as closely linked with genotype but more influenced by social factors such as family responsibilities and daily work routines. Another recently published study also suggested that the original classification criteria of Horne and Östberg (1976) are not useful for the classification of chronotype in middle-aged subjects (Paine et al., 2006). From age 50 years onwards, the allele frequencies of the extreme individuals appeared to exhibit a trend towards separating from the mean once again, although the difference was not significant. This observation may suggest that the influence of family and work schedules on the HÖ score begins to weaken, with people in this age group perhaps returning to a lifestyle more in keeping with their endogenous circadian phenotype. Indeed, it has previously been reported that the association between circadian period and entrained circadian phase was stronger in younger than in older subjects (Duffy and Czeisler, 2002), suggesting that these age-related changes in the strength of the associations with diurnal preference can also be observed at the physiological level.

The age-related change in allele frequency between younger (18–39 years of age) and older subjects suggests that the association between the PER3 polymorphism and diurnal preference is strongest in young people. This finding probably ought to be given consideration when selecting participants for diurnal preference studies with and without a genetic component, and when analysing the results. Differentiation by age group may also be a valuable tool for future genetic association studies of diurnal preference.

Acknowledgements

This article is dedicated to the memory of Sabiha Foster, who was instrumental in organising both of our studies at the Science Museum. The investigation was supported by the Wellcome Trust (grant number 074293). We thank Rebecca Richards and Emily Scott at the Science Museum for helping with the organisation of the project during Sabiha’s illness, Dion Chang for help with the computer based HÖ questionnaire and Jayshan Carpen, Lynette James, Aaron Jenkins, Claire Luke, Rob Meadows, Benita Middleton, Jennifer Sanders and Antoine Viola for assisting with the sample collection.

References

  1. Adan A, Natale V. Gender differences in morningness–eveningness preference. Chronobiol Int. 2002;19:709–720. doi: 10.1081/cbi-120005390. [DOI] [PubMed] [Google Scholar]
  2. Archer SN, Robilliard D, Skene DJ, Smits MG, Williams A, Arendt J, von Schantz M. A length polymorphism in the circadian clock gene PER3 is linked to delayed sleep phase syndrome and extreme diurnal preference. Sleep. 2003;26:413–415. doi: 10.1093/sleep/26.4.413. [DOI] [PubMed] [Google Scholar]
  3. Caci H, Mattei V, Bayle FJ, Nadalet L, Dossios C, Robert P, Boyer P. Impulsivity but not venturesomeness is related to morningness. Psychiatry Res. 2005;134:259–265. doi: 10.1016/j.psychres.2004.02.019. [DOI] [PubMed] [Google Scholar]
  4. Carrier J, Monk TH, Buysse DJ, Kupfer DJ. Sleep and morningness–eveningness in the ‘middle’ years of life (20–59 years) J Sleep Res. 1997;6:230–237. doi: 10.1111/j.1365-2869.1997.00230.x. [DOI] [PubMed] [Google Scholar]
  5. Dijk DJ, Duffy JF, Czeisler CA. Contribution of circadian physiology and sleep homeostasis to age-related changes in human sleep. Chronobiol Int. 2000;17:285–311. doi: 10.1081/cbi-100101049. [DOI] [PubMed] [Google Scholar]
  6. Duffy JF, Czeisler CA. Age-related change in the relationship between circadian period, circadian phase, and diurnal preference in humans. Neurosci Lett. 2002;318:117–120. doi: 10.1016/s0304-3940(01)02427-2. [DOI] [PubMed] [Google Scholar]
  7. Duffy JF, Dijk DJ, Hall EF, Czeisler CA. Relationship of endogenous circadian melatonin and temperature rhythms to self-reported preference for morning or evening activity in young and older people. J Investig Med. 1999;47:141–150. [PMC free article] [PubMed] [Google Scholar]
  8. Ebisawa T, Uchiyama M, Kajimura N, Mishima K, Kamei Y, Katoh M, Watanabe T, Sekimoto M, Shibui K, Kim K, Kudo Y, et al. Association of structural polymorphisms in the human period3 gene with delayed sleep phase syndrome. EMBO Rep. 2001;2:342–346. doi: 10.1093/embo-reports/kve070. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hardy GH. Mendelian proportions in a mixed population. Science. 1908;28:49–50. doi: 10.1126/science.28.706.49. [DOI] [PubMed] [Google Scholar]
  10. Horne JA, Östberg O. A self-assessment questionnaire to determine morningness–eveningness in human circadian rhythms. Int J Chronobiol. 1976;4:97–110. [PubMed] [Google Scholar]
  11. Hur YM, Bouchard TJ, Lykken DT. Genetic and environmental influence on morningness–eveningness. Pers Individ Dif. 1998;25:917–925. [Google Scholar]
  12. Jenkins A, Archer SN, von Schantz M. Expansion during primate radiation of a variable number tandem repeat in the coding region of the circadian clock gene Period3 . J Biol Rhythms. 2005;20:470–472. doi: 10.1177/0748730405278442. [DOI] [PubMed] [Google Scholar]
  13. Kerkhof GA, Van Dongen HP. Morning-type and eveningtype individuals differ in the phase position of their endogenous circadian oscillator. Neurosci Lett. 1996;218:153–156. doi: 10.1016/s0304-3940(96)13140-2. [DOI] [PubMed] [Google Scholar]
  14. Monk TH, Buysse DJ, Potts JM, DeGrazia JM, Kupfer DJ. Morningness–eveningness and lifestyle regularity. Chronobiol Int. 2004;21:435–443. doi: 10.1081/cbi-120038614. [DOI] [PubMed] [Google Scholar]
  15. Nadkarni NA, Weale ME, von Schantz M, Thomas MG. Evolution of a length polymorphism in the human PER3 gene, a component of the circadian system. J Biol Rhythms. 2005;20:490–499. doi: 10.1177/0748730405281332. [DOI] [PubMed] [Google Scholar]
  16. Paine SJ, Gander PH, Travier N. The epidemiology of morningness/eveningness: influence of age, gender, ethnicity, and socioeconomic factors in adults (30–49 years) J Biol Rhythms. 2006;21:68–76. doi: 10.1177/0748730405283154. [DOI] [PubMed] [Google Scholar]
  17. Pereira DS, Tufik S, Louzada FM, Benedito-Silva AA, Lopez AR, Lemos NA, Korczak AL, D’Ameida V, Pedrazzoli M. Association of the length polymorphism in the human PER3 gene with the delayed sleep-phase syndrome: does latitude have an influence upon it? Sleep. 2005;28:29–32. [PubMed] [Google Scholar]
  18. Robilliard D, Archer SN, Arendt J, Lockley SW, Hack LM, English J, Leger D, Smits MG, Williams A, Skene DJ, von Schantz M. The 3111Clock gene polymorphism is not associated with sleep and circadian rhythmicity in phenotypically characterized human subjects. J Sleep Res. 2002;11:305–312. doi: 10.1046/j.1365-2869.2002.00320.x. [DOI] [PubMed] [Google Scholar]
  19. Roenneberg T, Wirz-Justice A, Merrow M. Life between clocks: daily temporal patterns of human chronotypes. J Biol Rhythms. 2003;18:80–90. doi: 10.1177/0748730402239679. [DOI] [PubMed] [Google Scholar]
  20. Roenneberg T, Kuehnle T, Pramstaller PP, Ricken J, Havel M, Guth A, Merrow M. A marker for the end of adolescence. Curr Biol. 2004;14:R1038–R1039. doi: 10.1016/j.cub.2004.11.039. [DOI] [PubMed] [Google Scholar]
  21. Taillard J, Philip P, Coste O, Sagaspe P, Bioulac B. The circadian and homeostatic modulation of sleep pressure during wakefulness differs between morning and evening chronotypes. J Sleep Res. 2003;12:275–282. doi: 10.1046/j.0962-1105.2003.00369.x. [DOI] [PubMed] [Google Scholar]
  22. Taillard J, Philip P, Chastang JF, Bioulac B. Validation of Horne and Ostberg morningness–eveningness questionnaire in a middle-aged population of French workers. J Biol Rhythms. 2004;19:76–86. doi: 10.1177/0748730403259849. [DOI] [PubMed] [Google Scholar]
  23. Tankova I, Adan A, Buela-Casal G. Circadian typology and individual differences. A review. Pers Individ Dif. 1994;16:671–684. [Google Scholar]
  24. Vink JM, Groot AS, Kerkhof GA, Boomsma DI. Genetic analysis of morningness and eveningness. Chronobiol Int. 2001;18:809–822. doi: 10.1081/cbi-100107516. [DOI] [PubMed] [Google Scholar]

RESOURCES