Archer, SN

Arendt, J

Robilliard, DL

Skene, DJ

Smits, M

von Schantz, M

Williams, A

SLEEP

University of Surrey

INTRODUCTIONSLEEP TIMING AND STRUCTURE ARE STRONGLY INFLU-ENCED BY THE CIRCADIAN SYSTEM,1 which anticipates daylength and generates daily rhythms from a master pacemaker in thesuprachiasmatic nuclei.2 Every day, environmental photic time cues areprocessed via retinal input pathways to synchronize (entrain) the circa-dian pacemaker to the 24-hour day. In the absence of external time cues,the free-running endogenous circadian period τ is expressed. Diurnalpreference, as determined by the Horne-Östberg (HO) questionnaire3, avalidated quantitative tool, has been shown to correlate with τ.4 The rel-atively rare conditions known as advanced and delayed sleep phase syn-dromes (ASPS/DSPS) have been described as pathologic extremes ofdiurnal preference and may be linked to extremely short or long τ,respectively.5The accepted model for the molecular machinery that generates circa-dian rhythms involves a number of clock genes and their products.6 ThePeriod (Per) gene family is a central component in this mechanism, pro-viding negative auto-feedback on its own expression. Per transcripts andPER proteins oscillate with period lengths correlated to the observed τ.7Phosphorylation targets PER for degradation, imposing a rate-limitingstep on the amount of PER protein available for dimerization and subse-quent nuclear translocation. A mutation in Per2 has been reported toassociate with ASPS, potentially by disrupting a target site for phospho-rylation by casein kinase 1 (CK1) ε.  Here, we report a novel link between a length polymorphism in Per3and diurnal preference in humans. Homozygous Per3 knockout micedisplay a free-running τ 30 minutes shorter than the wildtype.8 Five Per3polymorphisms have been reported in a Japanese population, occurringin four haplotypes.9 One of these haplotypes was reported to be morefrequent in DSPS subjects, although the association between the fivepolymorphisms within this haplotype and the disorder were not deter-mined. Taking a different approach, we focused specifically on a length-polymorphic repeat region composed of either 4 or 5 units, which isdescribed, but not specifically analyzed, in the previous paper. Theprevalence of this polymorphism was studied both in subjects withextreme diurnal preference and in DSPS patients.METHODSOut of 484 volunteers who completed the Horne-Östberg question-naire and donated buccal DNA samples, the 7% of subjects with thehighest (morning preference) and lowest (evening preference) HOscores were selected, together with a control group of equal size with anintermediate HO score, as described in a previous report.10 Blood sam-ples were also collected from 16 unrelated patients (8 males, 8 females,aged 16-27 years) suffering from intrinsic DSPS, also described earli-er.10 Informed consent was obtained from all subjects after explanationof the nature of the study. The study was granted approval by the insti-tutional Advisory Committee on Ethics and followed the tenets of theDeclaration of Helsinki. Genotyping was performed using polymerasechain reaction with the primers described by Ebisawa et al 9 using theProofSprinter polymerase mixture (Hybaid, Ashford, Kent) and the fol-lowing amplification conditions: 94°C for 3 minutes, then 38 cycles of94°C for 45 seconds, 58° for 45 seconds, and 72° for 1 minute. Agarosegel electrophoresis was used to identify whether individuals were het-erozygous or homozygous for either of the Per3 repeat alleles.RESULTSFigure 1 shows the frequency of the 4- and 5-repeat alleles in groupswith extreme evening and extreme morning preference, as well as theintermediate group. A significant trend was observed between the threegroups (χ2 test for trend, P=0.030), with the frequency of the 5-repeatallele significantly higher in the morning-preference (5-repeat: 0.42, 4-repeat: 0.58) compared to the evening-preference group (5-repeat: 0.24,4-repeat: 0.76; Fisher’s Exact Test, P=0.047, odds ratio=2.205). In theDSPS patient group, the frequency of the 4-repeat allele was signifi-cantly higher (5-repeat: 0.12, 4-repeat: 0.88), compared to the totalLength Polymorphism in the Circadian Clock Gene Per3—Archer et alSLEEP, Vol. 26, No. 4, 2003 413A Length Polymorphism in the Circadian Clock Gene Per3 is Linked to DelayedSleep Phase Syndrome and Extreme Diurnal PreferenceCIRCADIAN RHYTHMSSimon N Archer, PhD1; Donna L Robilliard, BSc1; Debra J Skene, PhD1; Marcel Smits, MD, PhD2; Adrian Williams, MD3; Josephine Arendt, PhD, FRCPath1; Malcolmvon Schantz, PhD11Centre for Chronobiology, School of Biomedical and Life Science, University of Surrey, Guildford GU2 7XH, UK; 2Department of Neurology andSleep-Wake Disorders, Hospital de Gelderse Vallei, 6710 Ede, The Netherlands; 3Lane Fox Unit, St Thomas’ Hospital, Lambeth Palace Road, Lon-don SE1 7EH, UKStudy Objectives: To investigate the link between extreme diurnal pref-erence, delayed sleep phase syndrome, and a length polymorphism inPer3.Design: Subjects were genotyped using polymerase chain reaction.Patients or Participants: Subjects with defined diurnal preference asdetermined by the Horne-Östberg questionnaire and patients with delayedsleep phase syndrome.Measurements and Results: The Per3 polymorphism correlated signifi-cantly with extreme diurnal preference, the longer allele associating withmorningness and the shorter allele with eveningness. The shorter allelewas strongly associated with the delayed sleep phase syndrome patients,75% of whom were homozygous.Conclusion: The length of the Per3 repeat region identifies a potentialgenetic marker for extreme diurnal preference.Key Words: Circadian rhythms; phosphorylation; polymorphism (genet-ics); protein kinases; sleep disorder, circadian rhythmCitation:Archer SN, Robilliard DL, Skene DJ et al. A length polymorphismin the circadian clock gene Per3 is linked to delayed sleep phase syn-drome and extreme diurnal preference.  SLEEP 2003;26(4):413-415.Disclosure StatementThis research was supported by grants from the BBSRC (90/S10156 and90/C16668) and the MRC (G9901103).Submitted for publication December 2002Accepted for publication March 2003Address correspondence to: Dr. Malcolm von Schantz, Centre for Chronobiolo-gy, University of Surrey, Guildford GU2 7XH, UK; Tel: +44 1483 686468; Fax:+44 870 1334973; E-mail: m.von.schantz@surrey.ac.ukcontrol population (all 105 selected individuals; 5-repeat: 0.32, 4-repeat:0.68) (Fisher’s Exact Test, P=0.0224, odds ratio=3.352). No 5/5homozygotes were found in the DSPS group, and 75% were homozy-gous for the 4-repeat.DISCUSSIONThis is the first reported correlation between a polymorphism in aclock gene coding region and extreme diurnal preference in humans,including DSPS. The earlier publication by Ebisawa and coworkers9does not report this correlation in their material. This may be a reflectionof their study being based on carrier rather than allele frequencies, eth-nic differences, or both. Our findings provide some insights into thepotential function of Per3. CKIε phosphorylates all three PER proteins,regulating their stability and nuclear translocation.11 Each of the 4- or 5-repeat sequences in PER3 contain potential CK1ε phosphorylationmotifs clustered in a similar arrangement to those found in PER2 (Fig-ure 2).12 Of the amino acid residues in the repeat region, 32% are iden-tifiable as potential substrates for phosphorylation, as compared to 10%in the protein as a whole. Phosphorylation by CK1ε is enhanced byprephosphorylation of a lead serine or threonine residue in the recogni-tion motif. In PER2, a mutation in the first of a cluster of tandemlyarranged CK1ε recognition motifs has been hypothesized to reduce thechain of local phosphorylation leading to a more stabilized protein prod-uct and associated ASPS. In PER3, the decreased number of amino acidresidues available as phosphorylation substrates in the shorter variantwould predict a functional polymorphism in phosphorylation-dependenteffects. Because these sites are also arranged in a tandem array, themechanism may be very similar to that proposed in PER2. The identifi-cation of a robust link between this polymorphism and extreme diurnalpreference in humans indicates a precise way in which differential PER3phosphorylation may contribute to the phenotypic difference. This find-ing identifies a priority area for future studies and potential pharmaco-logic intervention. It also identifies a potential genetic marker forextreme diurnal preference, which may prove clinically useful in the dif-ferential diagnosis of DSPS. Because of the limited number of DSPSsufferers analyzed in this study, our finding should be confirmed in alarger cohort. It will also be of interest to investigate its prevalence inother sleep disorders, as well as its biochemical effects, including poten-tial differences in phosphorylation. The ability to tolerate night shiftwork, time zone transitions, and artificial time cues in a 24-hour societyis likely to depend upon the presence of specific clock gene variants,such as the one reported here.ACKNOWLEDGEMENTSWe are grateful to Dr. Derk-Jan Dijk for helpful suggestions and com-ments, and to Mrs. Sabiha Foster, Dr. Guy Warman, Mrs. Victoria War-man, Miss Hayley Tripp, Dr. Nelson Chong, and Dr. Shantha Wilson-Rajaratnam for assistance with material collection at the Science Muse-um.REFERENCES1. Dijk DJ, Czeisler CA. Contribution of the circadian pacemaker and the sleep homeostatto sleep propensity, sleep structure, electroencephalographic slow waves, and sleep spin-dle activity in humans.  J Neurosci 1995;15:3526-38.2. Weaver DR. The suprachiasmatic nucleus: a 25-year retrospective. J Biol Rhythms1998;13:100-12.3. Horne JA, Östberg O. A self-assessment questionnaire to determine morningness-eveningness in human circadian rhythms. Int J Chronobiol 1976;4:97-110.4. Duffy JF, Rimmer DW, Czeisler CA. Association of intrinsic circadian period withmorningness-eveningness, usual wake time, and circadian phase. Behav Neurosci2001;115:895-9.5. Jones CR, Campbell SS, Zone SE, et al. Familial advanced sleep-phase syndrome: ALength Polymorphism in the Circadian Clock Gene Per3—Archer et alSLEEP, Vol. 26, No. 4, 2003 414a b  Per3 allele frequencies  4 repeat 5 repeat Morning 0.58 0.42 Intermediate 0.68 0.32 Evening 0.76 0.24 Combined 0.68 0.32 DSPS 0.88 0.12  01020304050607080Morning Intermediate Evening DSPS4/44/55/5%   7 15 13 3 16 16 2 13 20 4 12 Figure 1—(a) Percentage of subjects with each of the Per3 repeat genotypes in the morn-ing-, intermediate- and evening-preference subject groups. Numbers are indicated above thebar for each genotype. (b) Frequency of alleles (N = 70) in morning-, intermediate- andevening-preference groups, in the 3 groups combined (N = 210), and in the delayed sleepphase syndrome (DSPS) group (N = 32). CK1PER4/5PASBPASAhPER3SPATTGALSTGSPPRENP SHPTASALSTGSPPMKNP SHPTASALSTGSPPMKNP SHPTASTLSMGLPPSRTP SHPTATVLSTGSPPSESPSPATTGALSTGSPPRENP SHPTASALSTGSPPMKNP ------------------ SHPTASTLSMGLPPSRTP SHPTATVLSTGSPPSESP1 2 3 4 55R4R1 130 270 973-1063 1210Figure 2—Schematic diagram of the human PER3 protein. Binding to other PER molecules occurs at the PAS A and B domains. The 5- and 4-repeat region amino acid sequences are shownexpanded below the sequence (5R and 4R, respectively). The repeats are numbered 1-5 and the one that is missing in the 4-repeat allele is indicated by dashes. Predicted targets for caseinkinase 1 (CK1) ε phosphorylation are indicated above by filled circles (the consensus CK1ε motif is Sp/Tp-X1-3-S/T, where the lead serine or threonine must be prephosphorylated, X is 1-3nonspecific spacing residues, and the target serine or threonine is underlined).short-period circadian rhythm variant in humans. Nat Med 1999;5:1062-5.6. Reppert SM, Weaver DR. Molecular analysis of mammalian circadian rhythms. AnnuRev Physiol 2001;63:647-76.7. Field MD, Maywood ES, O’Brien JA, Weaver DR, Reppert SM, Hastings MH. Analy-sis of clock proteins in mouse SCN demonstrates phylogenetic divergence of the circa-dian clockwork and resetting mechanisms. Neuron 2000;25:437-47.8. Shearman LP, Jin X, Lee C, Reppert SM, Weaver DR. Targeted disruption of the mPer3gene: subtle effects on circadian clock function. Mol Cell Biol 2000;20:6269-75.9. Ebisawa T, Uchiyama M, Kajimura N, et al. Association of structural polymorphisms inthe human period3 gene with delayed sleep phase syndrome. EMBO Rep 2001;2:342-6.10. Robilliard D, Archer SN, Arendt J, et al. The 3111Clock gene polymorphism is not asso-ciated with sleep and circadian rhythmicity in phenotypically characterized human sub-jects. J Sleep Res 2002;11:305-312.11. Akashi M, Tsuchiya Y, Yoshino T, Nishida E. Control of intracellular dynamics of mam-malian period proteins by casein kinase Iε (CKIε) and CKIδ in cultured cells. Mol CellBiol 2002;22:1693-703.12. Toh KL, Jones CR, He Y, et al. An hPer2 phosphorylation site mutation in familialadvanced sleep phase syndrome. Science 2001;291:1040-3.Length Polymorphism in the Circadian Clock Gene Per3—Archer et alSLEEP, Vol. 26, No. 4, 2003 415

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A length polymorphism in the circadian clock gene Per3 is linked to delayed sleep phase syndrome and extreme diurnal preference

A length polymorphism in the circadian clock gene Per3 is linked to delayed sleep phase syndrome and extreme diurnal preference

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