BMC Medical Genetics

BioMed Central

Research

Open Access

Genome-wide association of sleep and circadian phenotypes
Daniel J Gottlieb*1,2,3, George T O'Connor1,2 and Jemma B Wilk1,2
Address: 1The National Heart, Lung, and Blood Institute's Framingham Heart Study, Framingham, MA, USA, 2Boston University School of
Medicine, 715 Albany Street, Boston, MA 02118, USA and 3VA Boston Healthcare System, 1400 VFW Parkway, West Roxbury, MA 02130, USA
Email: Daniel J Gottlieb* - gottlieb@bu.edu; George T O'Connor - goconnor@lung.bumc.bu.edu; Jemma B Wilk - jwilk@bu.edu* Corresponding author

Published: 19 September 2007
<supplement>
S Vasan</editor><title><note>Research</note><p>The Framingham <url>http://www.biomedcentral.com/content/pdf/1471-2350-8-S1-info.pdf</url>
Heart Study 100,000 single nucleotide polymorphisms resource</p> </title> <editor>Emelia</supplement>
J Benjamin, Caroline S Fox, Daniel J Gottlieb, Cashell E Jaquish, Daniel Levy, James B Meigs, Christopher Newton-Cheh, Christopher J O'Donnell and Ramachandran

BMC Medical Genetics 2007, 8(Suppl 1):S9

doi:10.1186/1471-2350-8-S1-S9This article is available from: http://www.biomedcentral.com/1471-2350/8/S1/S9© 2007 Gottlieb et al; licensee BioMed Central Ltd.
This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract
Background: Numerous studies suggest genetic influences on sleepiness and circadian rhythms.
The Sleep Heart Health Study collected questionnaire data on sleep habits and sleepiness from
2848 Framingham Heart Study Offspring Cohort participants. More than 700 participants were
genotyped using the Affymetrix 100K SNP GeneChip, providing a unique opportunity to assess
genetic linkage and association of these traits.
Methods: Sleepiness (defined as the Epworth Sleepiness Scale score), usual bedtime and usual
sleep duration were assessed by self-completion questionnaire. Standardized residual measures
adjusted for age, sex and BMI were analyzed. Multipoint variance components linkage analysis was
performed. Association of SNPs to sleep phenotypes was analyzed with both population-based andfamily-based association tests, with analysis limited to 70,987 autosomal SNPs with minor allele
frequency ≥10%, call rate ≥80%, and no significant deviation from Hardy-Weinberg equilibrium (p≥ 0.001).
Results: Heritability of sleepiness was 0.29, bedtime 0.22, and sleep duration 0.17. Both genotype
and sleep phenotype data were available for 749 subjects. Linkage analysis revealed five linkage
peaks of LOD >2: four to usual bedtime, one to sleep duration. These peaks include several
candidate sleep-related genes, including CSNK2A2, encoding a known component of the circadianmolecular clock, and PROK2, encoding a putative transmitter of the behavioral circadian rhythm
from the suprachiasmatic nucleus. Association tests identified an association of usual bedtime witha non-synonymous coding SNP in NPSR1 that has been shown to encode a gain of function mutationof the neuropeptide S receptor, whose endogenous ligand is a potent promoter of wakefulness.
Each copy of the minor allele of this SNP was associated with a 15 minute later mean bedtime. The
lowest p value was for association of sleepiness with a SNP located in an intron of PDE4D, which
encodes a cAMP-specific phosphodiesterase widely expressed in human brain. Full association
results are posted at http://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?id=phs000007.
Conclusion: This analysis confirms prior reports of significant heritability of sleepiness, usual
bedtime, and usual sleep duration. Several genetic loci with suggestive linkage to these traits are
identified, including linkage peaks containing circadian clock-related genes. Association tests identify
NPSR1 and PDE4D as possible mediators of bedtime and sleepiness.

Page 1 of 8
(page number not for citation purposes)

BMC Medical Genetics 2007, 8(Suppl 1):S9

Background
Daytime sleepiness is a common symptom, experienced
at least 3 days per week by 29% of respondents in a recent
poll of U.S. adults [1]. Sleepiness is a major cause of
motor vehicle and occupational accidents, impaired
social function, and reduced quality of life. Within individuals, the level of sleepiness is modulated by a combination of homeostatic (duration of wakefulness) andcircadian (time of day) factors [2]. While behavioral factors and sleep disorders contribute to daytime sleepiness,there is great individual variability in the susceptibility tosleepiness in the context of disorders of sleep fragmentation [3] or sleep deprivation [4], which appears to be a stable individual trait. Evidence from several studies
indicates that excessive sleepiness is heritable, with heritability estimates from recent twin studies in the range of
0.38–0.48 [5-7]. Persistent circadian rhythm disorders,such as advanced or delayed sleep phase syndrome, arerelatively uncommon, estimated to affect <1% of the adult
population [8]. However, individual differences in diurnal preference (morning types or "larks" versus evening
types or "owls") have important implications for work
scheduling and performance that are highly relevant in an
economy in which almost one-fifth of employees areengaged in shift work [9]. Both twin and family studies
suggest that diurnal preference is heritable, with heritability estimates of 0.23–0.47 for usual bedtime or more formal assessment of diurnal preference [10-12]. Usual sleep
duration is an important determinant of daytime sleepiness; moreover, both short and long sleep duration havebeen associated in numerous epidemiologic studies withhypertension [13], diabetes mellitus [14,15], coronary
heart disease [16] and mortality [17-19], although themechanisms underlying these associations are poorly
understood. Significant heritability of usual sleep duration has been reported, with heritability estimates of
0.40–0.44 [10,20].
While sleepiness, diurnal preference, and sleep duration
have long been recognized as heritable traits, the genetic
basis of this heritability is largely unknown. While it has
been suggested that heritability of sleepiness may reflect
genetic influences on sleep-disordered breathing [5], sleep
drive is itself a highly regulated phenomenon and may be
influenced by variations in the numerous genes involvedin the circadian and homeostatic regulation of sleep andwakefulness. For example, a polymorphism in the geneencoding adenosine deaminase is reportedly associatedwith an increase in slow-wave sleep, a marker of homeostatic sleep drive [21]. Similarly, a polymorphism in
HCRTR2, the gene encoding the orexin/hypocretin receptor 2, has been identified in 2 patients with idiopathic
hypersomnolence but in no non-sleepy controls [22]. A
polymorphism in the gene encoding this receptor is
known to cause autosomal dominant canine narcolepsy

http://www.biomedcentral.com/1471-2350/8/S1/S9[23,24], although the role of polymorphisms in genes ofthe orexin/hypocretin system in human narcolepsy or
daytime sleepiness in general remains uncertain. Polymorphisms in the human period 2 (PER2) and casein
kinase 1d (CSNK1D) genes, known elements of the circadian molecular clock, are associated with autosomal dominant advanced sleep phase syndrome in isolated families[25,26]. A polymorphism in the 3'-untranslated region of
the CLOCK gene has been inconsistently reported in association with evening preference [27-29] and a length polymorphism in a tandem repeat region of the period 3
protein, containing either 4 or 5 repeats of an 18-aminoacid motif, is reportedly associated with diurnal preference [30]. These examples notwithstanding, it appears
that sleepiness and diurnal preference are polygenic traits.
In a genome-wide linkage analysis of C57BL/6J × BALB/cJ
hybrid mice, 14 loci were identified that were significantly
linked to circadian phenotypes; only one of these loci wasnear a gene proposed to be part of the core mammalian
circadian clock [31]. We are unaware of any publishedgenome-wide linkage or association studies of daytime
sleepiness, diurnal preference, or sleep duration inhumans.
The present study takes advantage of sleep phenotype datacollected by the Framingham Heart Study at the Offspring
Cycle 6 Examination. These data include measures of daytime sleepiness, usual bedtime, usual sleep duration, andsleep-disordered breathing. The aims of this study were to
replicate, in this unselected family-based sample, priorreports of heritability of these traits and to conduct
genome-wide linkage and association studies of these
traits. Although sleep phenotype data were collected fromfewer than half of all Offspring participants, heritability ofthe sleep-related phenotypes was confirmed in this sample and preliminary linkage and association studies identified several loci of interest.

Methods
Subjects of this study are drawn from the 2848 Framingham Offspring Study participants who completed sleep
habits questionnaires between 1995 and 1998 (Offspring
Examination Cycle 6) for the Sleep Heart Health Study, alongitudinal study of the cardiovascular consequences of
sleep-disordered breathing that has been described elsewhere [32]. Of these subjects, 891 members of 371 pedigrees had biological relatives with valid sleep phenotype
data and thus contributed to the heritability analyses.
Genome-wide SNP genotyping was performed in the"Family Plate Set" of 1345 members of the largest nuclear
families participating in the Framingham Original andOffspring cohorts using an Affymetrix 100K SNP GeneChip, as described in the Framingham Heart Study 100K
Project Overview [33]. A maximum of 738 members ofPage 2 of 8
(page number not for citation purposes)

BMC Medical Genetics 2007, 8(Suppl 1):S9

203 families contributed informative data to each of thegenetic association analyses of sleep phenotypes.
Data on daytime sleepiness, usual bedtime, and usual
sleep duration were obtained from a self-completion
questionnaire either handed to the participant at the timeof a regularly scheduled visit to the Framingham Study
clinic or mailed to the participant. Sleepiness was definedas the score on the Epworth Sleepiness Scale, a widelyused and well validated 8-item questionnaire that asks thelikelihood of falling asleep in a variety of commonly
encountered situations [34,35]. Usual bedtime wasobtained from the single question, "What time to you
usually go to bed on weeknights (or work nights)?" Usual
sleep duration was obtained from the single question,"How many hours of sleep do you usually get on weeknights (or work nights)?" with integer response options.
The full Sleep Habits Questionnaire is available from the
Sleep Heart Health Study website [36]. Data on work shift
and retirement status were not available in this cohort;therefore, in order to exclude subjects in whom night shift
work might lead to spurious estimates of circadian phenotype, the 0.5% of subjects reporting a usual bedtime
between 5 AM and 6 PM were excluded from analyses ofusual bedtime and usual sleep duration. Those whose
usual bedtime differed by more than two hours between
weekdays and weekends (1.3%) were excluded from analyses of usual bedtime, as behavioral factors were presumed to have a major influence on this measure.
Similarly, those whose usual sleep duration differed bymore than two hours between weekdays and weekends(4.2%) were excluded from analyses of sleepiness andusual sleep duration. As sleepiness, bedtime, and sleep
duration may be influenced by age, sex and BMI[5,10,20], adjustment for these variables was made by linear regression. Standardized residuals of the adjusted
sleep phenotype variables were used in genetic analyses.
As further adjustment of sleepiness for usual sleep duration or self-reported symptoms of sleep-disorderedbreathing (snoring, nocturnal breathing pauses) reduced
sample size but had little impact on the linkage and association results, analyses using these further adjustments
are not presented. Standardized residuals were obtainedusing PROC REG in the SAS statistical software package(SAS version 9.1, SAS Institute, Cary, NC).
Heritability, linkage and genetic association analysis was
performed as described in the Framingham Heart Study
100K Project Overview [33]. Briefly, multipoint linkage
analysis was implemented using Merlin identity-bydescent estimates and variance component linkage in
SOLAR with a subset of 10,588 SNPs supplementing 612
microsatellite markers from a previous genome scan.
Association of SNPs to sleep phenotypes was studied withboth population-based association tests using generalized

http://www.biomedcentral.com/1471-2350/8/S1/S9

estimating equations (GEE) and family-based association
tests (FBAT). All association tests employed an additivemodel. Reported association test results are limited to
SNPs located on autosomes and meeting the following
quality control criteria: minor allele frequency of ≥10%;
call rate ≥80%; and no significant deviation from HardyWeinberg equilibrium (p ≥ 0.001). Of the 70,987 SNPs
meeting these criteria, 40,249 were located within 60 kb
of a known or putative gene. Physical locations are basedon National Center for Biotechnology Information build
35.
Of the 2848 Offspring Study participants who completed
the sleep habits questionnaire, 699 also underwent overnight polysomnography; therefore, polysomnographic
data on sleep-disordered breathing were available in asmall subset of subjects included in the Family Plate Set (n= 219). There were too few subjects to permit linkage analysis and, as power was very low for association studies,
these data are not considered in this manuscript but are
posted on the web site. The correlation of sleepiness, bedtime, and sleep duration with polysomnographicallymeasured apnea-hypopnea index was weak (correlation
coefficients -0.07 to 0.10).

Results
Sleep characteristics of the 749 subjects included in the
Framingham Study Family Plate Set with available sleep
phenotype data are shown in Table 1. Mean age was 55.8
(SD 9.4) years, mean BMI was 28.2 (SD 5.5) kg/m2, and51.5% were women. Heritability of sleepiness was 0.29 (p< 0.001), of usual bedtime 0.22 (p < 0.01), and of usual
sleep duration 0.17 (p = .02). The estimated heritability ofsleepiness was not appreciably reduced by further adjustment for usual sleep duration and the presence of habitualsnoring or witnessed apneas (0.28, p = 0.002). Linkage
analysis to these three phenotypes identified no linkage
peaks with LOD >3. Five linkage peaks with LOD >2 wereidentified, four to usual bedtime and one to usual sleep
duration (Table 2c). The region of the strongest of these, a
LOD score of 2.45 for usual bedtime with a linkage peakat 55.5 Mb on chromosome 16, includes the gene encoding casein kinase 2a2 (CSNK2A2, at 56.7 Mb), a known
element of the Drosophila circadian clock. The linkage
peak to usual sleep duration at 71.3 Mb on chromosome
3 includes the gene encoding prokineticin 2 (PROK2, at
71.9 Mb), which may be an important output moleculefrom the suprachiasmatic nucleus. Three additional linkage peaks to usual bedtime had maximum LOD scores of
1.9. One of these, located on chromosome 2, includes theinterleukin-1 cytokine cluster. Another, located on chromosome 4 at 62.5 Mb, includes the CLOCK gene (at 56.0–
56.1 Mb). A smaller linkage peak of LOD 1.5 was noted inthis region (at 57.1 Mb) for sleepiness. Kurtosis was low
for sleepiness and usual bedtime, at 0.2 and 1.1, respec-

Page 3 of 8
(page number not for citation purposes)

BMC Medical Genetics 2007, 8(Suppl 1):S9

http://www.biomedcentral.com/1471-2350/8/S1/S9

tively, although kurtosis of usual sleep duration was highat 3.0.
The five autosomal SNPs with the lowest p-value for association for each sleep phenotype by population-based andfamily-based association tests, and meeting quality control standards, are displayed in Tables 2a and 2b. Only
one of these is located in a coding region: rs324981 in
NPSR1, associated with usual bedtime, whose minor
allele (frequency 0.44) is a non-synonymous mutation
encoding an Asn107→Ile107 substitution in an exoloop lining the putative ligand-binding pocket of the neuropeptide S receptor [37]. The effect of this polymorphism isadditive, with adjusted mean bedtime delayed by 14.9
minutes in heterozygotes and 29.5 minutes in homozygotes. Only eight SNPs were associated with any of thesleep phenotypes with p < 10-5. The SNP with the lowest
p value for association to any of the sleep phenotypes isfor the association of rs1823068 with sleepiness by population-based association testing (p = 2.5*10-8). This SNP
is located in an intron of the gene encoding phosphodiesterase 4D (PDE4D). Other SNPs associated at p < 10-5
include one located in an intron of the gene encoding eyesabsent 1 (EYA1) and two within an intron of the geneencoding myosin VIIA and Rab interacting protein(MYRIP) identified by population-based association tests,
and two in or near the gene encoding opioid binding protein/cell adhesion molecule (OPCML) by family-based
association tests. One was not located near a known gene.
The results of population-based and family-based association tests were modestly correlated. For example, when
SNPs were ranked based on the p value for association
with sleepiness, the Spearman correlation coefficient for
rankings from population-based versus family-based tests
was 0.22. Tables 2a and 2b show p-values for both
approaches, allowing assessment of concordance acrossapproaches for these SNPs. Only 3 SNPs were associatedwith sleepiness at p < 0.001 by both approaches; thesewere located in FHIT, VTA1, and LRP1B. Of the 5 SNPs
meeting this criterion for usual bedtime, none was in ornear a known gene, and no SNPs met this criterion for
usual sleep duration. Similarly, little overlap was seen
across the three phenotypes. No SNPs associated withsleepiness at p < 0.001 were associated with either usual
bedtime or sleep duration at this nominal significancelevel for either population-based or family-based tests,
and only 4 SNPs met this criterion for overlap between
usual bedtime and sleep duration; three were located in
known genes RYR2, JAZF1, and NDRG1.
Among potential candidate genes for sleep-related phenotypes, many were poorly represented on the Affymetrix
100K GeneChip. No SNP meeting quality control standards was located within PROK2, identified in the linkage
analysis as a possible candidate for usual sleep duration.
Only a single SNP was located in PER2 and none in
CSNK1D or PER3, genes associated with familial
advanced or delayed sleep phase syndromes. In contrast,
10 SNPs meeting quality control standards were typed
within the CLOCK gene and 5 within CSNK2A2, none ofwhich was significantly associated with any sleep phenotype by either the population-based or family-based
approaches (lowest p value 0.12). The full results of thegenetic association studies are available at the National
Center for Biotechnology Information dbGaP website[38].

Discussion
In this family-based study, we have confirmed significant
heritability of sleepiness, usual bedtime, and usual sleep
duration that had been previously reported primarily
from twin cohorts [5-7,10-12,20]. The heritability estimates in this study are lower than those reported in the literature from twin studies. This may reflect a greater
contribution of environmental influences on these phenotypes in the present study that is detected as correlation
between spouse-pairs. Alternatively, an underestimate of
the shared environmental variance in twin studies may
cause them to overstate the genetic contribution, as theestimated heritability of usual bedtime in this study issimilar to that for diurnal preference in a previous familybased study [12]. We are unaware of any prior genomewide linkage or association studies of these sleep-related
phenotypes in humans. Because sleep phenotypes wereavailable from only 56% of subjects included in the Framingham Family Plate Set, study power was limited and nolinkage peaks reaching conventional levels of genome-

Table 1: Sleep phenotypic characteristics of Family Plate Set subjects
PhenotypeN

Offspring Exam
cycle

Unadjusted Mean(SD)

Adjustment variables

Heritability (SE)

Webpost name*

Epworth Sleepiness Scale score
Usual bedtime, (hr:min)
Usual sleep duration, hrs

721
738
736

6
6
6

6.4 (3.8)
10:50 PM (1:06)
7.28 (1.06)

age, sex, BMI
age, sex, BMI
age, sex, BMI

0.29 (0.09)
0.22 (0.08)
0.17 (0.08)

essresid1
bedtimeresid
sleepdurresid*The age-, sex- and BMI-adjusted residual apnea-hypopnea index (rdiresid) is also posted in the on-line repository, although it is not discussed in this manuscript. Also posted
in the on-line repository but not included in this manuscript are the unadjusted Epworth Sleepiness Scale score (ess), usual bedtime (bedtime), and usual sleep duration(sleepdur), as well as Epworth Sleepiness Scale score additionally adjusted for usual sleep duration (essresid2) and for both usual sleep duration and the presence of selfreported sleep-disordered breathing symptoms of snoring three or more nights per week or any witnessed nocturnal apneas (essresid3).

Page 4 of 8
(page number not for citation purposes)

BMC Medical Genetics 2007, 8(Suppl 1):S9

http://www.biomedcentral.com/1471-2350/8/S1/S9

Table 2: Top genetic association and linkage results
2a – Top association results from population-based association tests
Phenotype

SNP

Sleepiness
Sleepiness
Sleepiness
Sleepiness
Sleepiness
BedtimeBedtimeBedtimeBedtimeBedtimeSleep duration
Sleep duration
Sleep duration
Sleep duration
Sleep duration

rs1823068
rs2218488
rs2247614
rs7073579
rs9298693
rs949175
rs2288292
rs324981
rs10483871
rs10507551
rs6599077*
rs10492604
rs10489832
rs2256551
rs2165207

Chr

Physical Position

5
8
21
10
9
11
11
7
14
13
3
13
1
20
12

58,711,806
72,426,510
35,553,557
1,364,007
13,891,409
97,242,217
12,452,275
34,591,353
75,141,034
46,659,884
40,071,622
57,802,314
155,267,429
51,066,908
72,418,535

GEE p-value

FBAT p-value

2.5*10-8
3.2*10-6.000011.000014.000016
9.7*10-6.000017.000018.000045.000048
1.4*10-7
4.2*10-6.000028.000065.000011

.069
.070
.0045
.0081
.28
.38
.0096
.089
.0065
.032
.020
.038
.0043
.11
.13

GEE p-value

FBAT p-value.00054.53.0024.000096
.013
.0026
.33
.031
.37
.0099
.85
.059
.13
.037
.20

.000019.000019.000024.000056.0000646.1*10-6.000019.000022.000025.000035.000072.00011.00014.00021.00024

Gene Region
PDE4D
EYA1
ADARB2PARVA
NPSR1
C14orf58
MYRIP
OR10K1, OR10K2
TSHZ2

2b – Top association results from family-based association tests
Phenotype

SNP

Sleepiness
Sleepiness
Sleepiness
Sleepiness
Sleepiness
BedtimeBedtimeBedtimeBedtimeBedtimeSleep duration
Sleep duration
Sleep duration
Sleep duration
Sleep duration

rs10510835
rs2189829
rs434052*
rs4896580
rs10520010
rs1940013*
rs2525724
rs1725021
rs932650
rs2985334
rs481233
rs2359894
rs2061579
rs10492507
rs6974138

Chr

Physical Position

3
7
14
6
4
11
7
7
10
1
6
18
12
13
7

60,222,161
49,387,124
35,613,399
142,518,529
150,482,987
131,786,861
120,203,894
136,350,575
115,337,349
29,165,643
126207158
40309270
73224522
48722291
74708138

Gene Region
FHIT

VTA1
OPCML
ING3
PTN
HABP2
EPB41
NCOA7

CDADC12c – Linkage peaks with LOD score >2.0
Phenotype

SNP closest to
linkage peakChrPhysical location of
linkage peak

Maximum LOD score

1.5 LOD support interval (Mb)

BedtimeBedtimeBedtimeBedtimers28168
rs10498313
rs10503857
rs10512058

16
14
8
9

55,508,777
29,468,627
29,909,250
76,736,108

2.45
2.33
2.26
2.11

48.5 – 62.0
28.7 – 32.6
21.9 – 37.1
10.6 – 107.4*A second strongly associated SNP in this gene region is not reported in the table because it is in close proximity to and in linkage disequilibrium(D' 1.0) with the reported SNP.

wide significance were observed. Despite the greater heritability of sleepiness, most of the suggestive linkage peaks
observed in this study were linked to usual bedtime andto a lesser extent to usual sleep duration. Several of thesesuggestive linkage peaks contain genes of potential importance to the circadian molecular clock. Two of these peaks
are of particular interest. The linkage to usual bedtime on
chromosome 16 was the strongest observed in this study(LOD = 2.45), with a peak close to the gene CSNK2A2. Its
product, a catalytic subunit of casein kinase 2, has beenshown to be an important component of the circadianmolecular clock in Drosophila and other organisms [39].
Phosphorylation by casein kinase 2 promotes nuclear
translocation of the PERIOD gene product, and mutations
that impair catalytic activity or subunit multimerization
cause a lengthening of the circadian period [40,41].
Although casein kinase 2 has not previously been implicated in human circadian rhythm disorders, mutations in
the human genes encoding casein kinase 1d and period 2
are associated with familial advanced sleep phase syn-Page 5 of 8
(page number not for citation purposes)

BMC Medical Genetics 2007, 8(Suppl 1):S9

drome [25,26]. The linkage to usual sleep duration on
chromosome 3 (LOD = 2.17) has a peak close to the gene
PROK2. Its product is the precursor of prokineticin 2,
which is highly expressed in the suprachiasmatic nucleus,
regulated by the circadian molecular clock, and believed
to be an important output molecule from the suprachiasmatic nucleus, coordinating and transmitting the behavioral circadian rhythm to multiple brain regions [42,43].
Although not previously implicated in human disorders,
the total sleep duration of prokineticin null mice isreduced by 83.5 minutes per 24 hour period compared totheir wild-type littermates [44]. The modest linkage toboth bedtime and sleepiness near the CLOCK gene, a central component of the molecular circadian clock, is also
intriguing. PROK2 could not be evaluated in association
tests, as no SNPs were typed within this gene. As none of
the 5 SNPs within CSNK2A2 or the 10 within CLOCK was
significantly associated with usual bedtime, other genes inthese regions or chance may be responsible for these linkage peaks.
Association tests identified several loci that merit followup in other cohorts. The most interesting of these is anassociation of usual bedtime with a non-synonymous
coding SNP in NPSR1, which causes an Asn107→Ile107
substitution in the putative ligand-binding pocket of theneuropeptide S receptor [37]. This same variant has been
linked to asthma in several Caucasian populations[45,46] but has not been previously reported in association with any sleep or circadian phenotype. In mice, neuropeptide S is localized to a small area adjacent to thenoradrenergic locus ceruleus and its intraventricularadministration is a potent, transient stimulus to wakefulness [47]. The Asn107→Ile107 variant of the neuropeptide S
receptor is a gain of function mutation, increasing sensitivity of the receptor to neuropeptide S [37]. Consistent
with this effect on receptor function, mean bedtime is 15
minutes later for each copy of the gene encoding theAsn107→Ile107 variant.
Although not located in a coding region, the strong association of sleepiness with a SNP located in an intron of thegene encoding phosphodiesterase 4D also merits further
study. Phosphodiesterase 4 is a cAMP-specific phosphodiesterase that has multiple splice variants, with PDE4D
being widely expressed in human brain [48]. Mutations of
PDE4D have been associated with stroke risk in several
populations, possibly related to the role of PDE4 in modulating inflammatory processes, although the causal
nature of the association remains controversial [49,50].
While the nonselective phosphodiesterase inhibitors caffeine and theophylline have long been recognized to promote wakefulness, this is likely due to antagonism of
dopamine receptors rather than phosphodiesterase inhibition [51]. However, variation in the effects of PDE4D on

http://www.biomedcentral.com/1471-2350/8/S1/S9

brain intracellular levels of cAMP or extracellular levels of
adenosine might influence sleepiness, and the selective
PDE4 inhibitor rolipram is a weak promoter of wakefulness in rats [52].
This study has a number of limitations. The sleep phenotypes were assessed by questionnaire only. While the
Epworth Sleepiness Scale is a well-validated measure ofusual sleepiness, the single questions regarding usual bedtime and sleep duration provide only crude measures ofcircadian phenotype. Moreover, as subjects did not have aclinical sleep evaluation, it was not possible to control forsleep apnea or other primary sleep disorders. A sleepinessphenotype further adjusted for self-reported usual sleep
duration and frequent snoring or witnessed apneas wasanalyzed, however, and results are included in the web
repository. This gave results very similar to those presented in this manuscript, although with somewhat lower
power due to an additional 15% missing phenotype data.
The present study has statistical limitations as well. The
relatively small number of subjects included in the analysis limits the power to detect true linkage and association,
while the large number of SNPs tested and apparent inflation of Type I error rates for the population-based association tests makes it likely that many of the observed
associations are false positive findings. Kurtosis of themeasure of sleep duration may have inflated the LOD
scores from linkage analysis. Thus, all results of this study
require replication in other populations. These statisticallimitations are discussed in more detail in the Overview[33].
Notwithstanding these limitations, this study begins toapply the powerful methodologies of genetic epidemiology to the study of common sleep and circadian phenotypes, and identifies for further study several genes thathave not previously been implicated in human sleep andcircadian disorders. These findings require replication,
which will be pursued in other cohorts and by a planned
expansion of the SNP genotyping using a 500,000 SNP
gene chip in a substantially larger sample of Framingham
Study subjects. Collection of more detailed sleep phenotype data from a larger sample of Framingham Study subjects will increase the power to detect novel genes
influencing sleep and circadian phenotypes.

Conclusion
This analysis confirms, in a family-based sample, priorreports of significant heritability of sleepiness, usual bedtime, and usual sleep duration. It identifies several genetic
loci with suggestive linkage to these traits, including linkage peaks containing the circadian clock-related genes
CSNK2A2, PROK2 and CLOCK. Among genes identified
by association tests as possible mediators of sleep and circadian phenotypes, those most promising based on the

Page 6 of 8
(page number not for citation purposes)

BMC Medical Genetics 2007, 8(Suppl 1):S9

strength of the associations and their known biologicalactivity are NPSR1 and PDE4D, which may influence
usual bedtime and daytime sleepiness, respectively. Whilethese findings require replication in other samples, they
provide evidence of the possible utility of genetic epidemiology approaches to understanding population variation in sleep and circadian phenotypes.

http://www.biomedcentral.com/1471-2350/8/S1/S9

10.
11.
12.

13.

Competing interests
The authors declare that they have no competing interests.

14.

Authors' contributions
All authors participated in the conception and design ofthe study. DJG and GTO participated in phenotype datacollection. DJG and JBW participated in the statisticalanalysis. DJG drafted the manuscript, with critical feedback from GTO and JBW. All authors read and approved
the final manuscript.

Acknowledgements
The authors wish to acknowledge the contribution of the Framingham
Study participants to this research. This research was supported by the
National Heart, Lung and Blood Institute through contract N01-HC 25195(Framingham Heart Study) and cooperative agreement U01 HL53941(Sleep Heart Health Study). A portion of the research was conducted using
the Boston University Linux Cluster for Genetic Analysis (LinGA) fundedby the NIH NCRR (National Center for Research Resources) Shared
Instrumentation grant (1S10RR163736-01A1). Dr. Wilk is supported by ayoung clinical scientist award from the Flight Attendant Medical ResearchInstitute.

15.

16.
17.
18.
19.
20.
21.

22.
This article has been published as part of BMC Medical Genetics Volume 8
Supplement 1, 2007: The Framingham Heart Study 100,000 single nucleotide polymorphisms resource. The full contents of the supplement areavailable online at http://www.biomedcentral.com/1471-2350/8?issue=S1.
23.

References
1.
2.
3.
4.

5.

6.
7.
8.
9.

National Sleep Foundation: 2005 Sleep in America Poll. Washington, DC
2005.
Dijk DJ, Duffy JF, Czeisler CA: Circadian and sleep/wake dependent aspects of subjective alertness and cognitive performance. J Sleep Res 1992, 1:112-117.
Kapur VK, Baldwin CM, Resnick HE, Gottlieb DJ, Nieto FJ: Sleepiness in patients with moderate to severe sleep-disorderedbreathing. Sleep 2005, 28:472-477.
Van Dongen HP, Baynard MD, Maislin G, Dinges DF: Systematicinterindividual differences in neurobehavioral impairment
from sleep loss: evidence of trait-like differential vulnerability. Sleep 2004, 27:423-433.
Carmelli D, Bliwise DL, Swan GE, Reed T: A genetic analysis of the
Epworth Sleepiness Scale in 1560 World War II male veteran twins in the NAS-NRC Twin Registry. J Sleep Res 2001,
10:53-58.
Desai AV, Cherkas LF, Spector TD, Williams AJ: Genetic influences
in self-reported symptoms of obstructive sleep apnoea andrestless legs: a twin study. Twin Res 2004, 7:589-595.
Watson NF, Goldberg J, Arguelles L, Buchwald D: Genetic andenvironmental influences on insomnia, daytime sleepiness,and obesity in twins. Sleep 2006, 29:645-649.
Ando K, Kripke DF, Ancoli-Israel S: Estimated prevalence ofdelayed and advanced sleep phase syndromes. Sleep Res 1995,
24:509.
Drake CL, Roehrs T, Richardson G, Walsh JK, Roth T: Shift work
sleep disorder: prevalence and consequences beyond that ofsymptomatic day workers. Sleep 2004, 27:1453-1462.

24.

25.
26.

27.
28.

29.

30.

Heath AC, Kendler KS, Eaves LJ, Martin NG: Evidence for genetic
influences on sleep disturbance and sleep pattern in twins.
Sleep 1990, 13:318-335.
Vink JM, Groot AS, Kerkhof GA, Boomsma DI: Genetic analysis ofmorningness and eveningness. Chronobiol Int 2001, 18:809-822.
Klei L, Reitz P, Miller M, Wood J, Maendel S, Gross D, Waldner T,
Eaton J, Monk TH, Nimgaonkar VL: Heritability of morningnesseveningness and self-report sleep measures in a family-based
sample of 521 hutterites. Chronobiol Int 2005, 22:1041-1054.
Gottlieb DJ, Redline S, Nieto FJ, Baldwin CM, Newman AB, Resnick
HE, Punjabi NM: Association of usual sleep duration withhypertension: the Sleep Heart Health Study. Sleep 2006,
29:1009-1014.
Ayas NT, White DP, Al-Delaimy WK, Manson JE, Stampfer MJ,
Speizer FE, Patel S, Hu FB: A prospective study of self-reported
sleep duration and incident diabetes in women. Diabetes Care
2003, 26:380-384.
Gottlieb DJ, Punjabi NM, Newman AB, Resnick HE, Redline S, Baldwin
CM, Nieto FJ: Association of sleep time with diabetes mellitus
and impaired glucose tolerance. Arch Intern Med 2005,
165:863-867.
Ayas NT, White DP, Manson JE, Stampfer MJ, Speizer FE, Malhotra A,
Hu FB: A prospective study of sleep duration and coronary
heart disease in women. Arch Intern Med 2003, 163:205-209.
Hammond EC: Some Preliminary Findings on Physical Complaints from a Prospective Study of 1,064,004 Men and
Women. Am J Public Health Nations Health 1964, 54:11-23.
Wingard DL, Berkman LF, Brand RJ: A multivariate analysis ofhealth-related practices: a nine-year mortality follow-up of
the Alameda County Study. Am J Epidemiol 1982, 116:765-775.
Kripke DF, Garfinkel L, Wingard DL, Klauber MR, Marler MR: Mortality associated with sleep duration and insomnia. Arch Gen
Psychiatry 2002, 59:131-136.
Partinen M, Kaprio J, Koskenvuo M, Putkonen P, Langinvainio H:
Genetic and environmental determination of human sleep.
Sleep 1983, 6:179-185.
Retey JV, Adam M, Honegger E, Khatami R, Luhmann UF, Jung HH,
Berger W, Landolt HP: A functional genetic variation of adenosine deaminase affects the duration and intensity of deep
sleep in humans. Proc Natl Acad Sci USA 2005, 102:15676-15681.
Thompson MD, Comings DE, bu-Ghazalah R, Jereseh Y, Lin L, WadeJ, Sakurai T, Tokita S, Yoshida T, Tanaka H, Yanagisawa M, Burnham
WM, Moldofsky H: Variants of the orexin2/hcrt2 receptor gene
identified in patients with excessive daytime sleepiness andpatients with Tourette's syndrome comorbidity. Am J Med
Genet B Neuropsychiatr Genet 2004, 129:69-75.
Lin L, Faraco J, Li R, Kadotani H, Rogers W, Lin X, Qiu X, de Jong PJ,
Nishino S, Mignot E: The sleep disorder canine narcolepsy iscaused by a mutation in the hypocretin (orexin) receptor 2
gene. Cell 1999, 98:365-376.
Chemelli RM, Willie JT, Sinton CM, Elmquist JK, Scammell T, Lee C,
Richardson JA, Williams SC, Xiong Y, Kisanuki Y, Fitch TE, Nakazato
M, Hammer RE, Saper CB, Yanagisawa M: Narcolepsy in orexin
knockout mice: molecular genetics of sleep regulation. Cell
1999, 98:437-451.
Toh KL, Jones CR, He Y, Eide EJ, Hinz WA, Virshup DM, Ptacek LJ,
Fu YH: An hPer2 phosphorylation site mutation in familial
advanced sleep phase syndrome. Science 2001, 291:1040-1043.
Xu Y, Padiath QS, Shapiro RE, Jones CR, Wu SC, Saigoh N, Saigoh K,
Ptacek LJ, Fu YH: Functional consequences of a CKIdelta mutation causing familial advanced sleep phase syndrome. Nature
2005, 434:640-644.
Katzenberg D, Young T, Finn L, Lin L, King DP, Takahashi JS, Mignot
E: A CLOCK polymorphism associated with human diurnal
preference. Sleep 1998, 21:569-576.
Robilliard DL, Archer SN, Arendt J, Lockley SW, Hack LM, English J,
Leger D, Smits MG, Williams A, Skene DJ, von Schantz M: The 3111
Clock gene polymorphism is not associated with sleep andcircadian rhythmicity in phenotypically characterized
human subjects. J Sleep Res 2002, 11:305-312.
Mishima K, Tozawa T, Satoh K, Saitoh H, Mishima Y: The 3111T/C
polymorphism of hClock is associated with evening preference and delayed sleep timing in a Japanese population sample. Am J Med Genet B Neuropsychiatr Genet 2005, 133:101-104.
Archer SN, Robilliard DL, Skene DJ, Smits M, Williams A, Arendt J,
von Schantz M: A length polymorphism in the circadian clock

Page 7 of 8
(page number not for citation purposes)

BMC Medical Genetics 2007, 8(Suppl 1):S9

31.

32.

33.

34.
35.
36.
37.

38.
39.
40.
41.
42.

43.
44.
45.

46.

47.

48.

49.

gene Per3 is linked to delayed sleep phase syndrome andextreme diurnal preference. Sleep 2003, 26:413-415.
Shimomura K, Low-Zeddies SS, King DP, Steeves TD, Whiteley A,
Kushla J, Zemenides PD, Lin A, Vitaterna MH, Churchill GA, Takahashi JS: Genome-wide epistatic interaction analysis reveals
complex genetic determinants of circadian behavior in mice.
Genome Res 2001, 11:959-980.
Quan SF, Howard BV, Iber C, Kiley JP, Nieto FJ, O'Connor GT, Rapoport DM, Redline S, Robbins J, Samet JM, Wahl PW: The Sleep
Heart Health Study: design, rationale, and methods. Sleep
1997, 20:1077-1085.
Cupples LA, Benjamin EJ, D'Agostino RB Sr, Demissie S, DeStefano
AL, Dupuis J, Falls K, Fox CS, Gottlieb DJ, Govindaraju DR, HeardCosta N, Hwang SJ, Kathiresan S, Kiel DP, Laramie JM, Larson MG,
Levy D, Lunetta KL, Mailman MD, Manning AK, Meigs JB, Murabito JM,
Newton-Cheh C, O'Connor GT, O'Donnell CJ, Pandey MA, Seshadri
S, Vasan RS, Wilk JB, Wolf PA, Yang Q, Atwood LD: The Framingham Heart Study 100K SNP genome-wide association study
resource: Overview of 17 phenotype working group reports.
BMC Med Genet 2007, 8(Suppl 1):S1.
Johns MW: A new method for measuring daytime sleepiness:
the Epworth sleepiness scale. Sleep 1991, 14:540-545.
Johns MW: Reliability and factor analysis of the Epworth
Sleepiness Scale. Sleep 1992, 15:376-381.
Sleep Heart Health Study Sleep Habits Questionnaire [http://www.jhucct.com/shhs/details/manual/forms/sh/shhshq2.pdf]
Reinscheid RK, Xu YL, Okamura N, Zeng J, Chung S, Pai R, Wang Z,
Civelli O: Pharmacological characterization of human andmurine neuropeptide s receptor variants. J Pharmacol Exp Ther
2005, 315:1338-1345.
National Center for Biotechnology Information dbGaP website[http://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?id=phs000007]
Lin JM, Kilman VL, Keegan K, Paddock B, Emery-Le M, Rosbash M,
Allada R: A role for casein kinase 2alpha in the Drosophila circadian clock. Nature 2002, 420:816-820.
Lin JM, Schroeder A, Allada R: In vivo circadian function of casein
kinase 2 phosphorylation sites in Drosophila PERIOD. J Neurosci 2005, 25:11175-11183.
Akten B, Jauch E, Genova GK, Kim EY, Edery I, Raabe T, Jackson FR:
A role for CK2 in the Drosophila circadian oscillator. Nat
Neurosci 2003, 6:251-257.
Cheng MY, Bullock CM, Li C, Lee AG, Bermak JC, Belluzzi J, Weaver
DR, Leslie FM, Zhou QY: Prokineticin 2 transmits the behavioural circadian rhythm of the suprachiasmatic nucleus.
Nature 2002, 417:405-410.
Li JD, Hu WP, Boehmer L, Cheng MY, Lee AG, Jilek A, Siegel JM, Zhou
QY: Attenuated circadian rhythms in mice lacking the prokineticin 2 gene. J Neurosci 2006, 26:11615-11623.
Hu WP, Li JD, Zhang C, Boehmer L, Siegel JM, Zhou QY: Alteredcircadian and homeostatic sleep regulation in prokineticin 2deficient mice. Sleep 2007, 30:247-256.
Laitinen T, Polvi A, Rydman P, Vendelin J, Pulkkinen V, Salmikangas P,
Makela S, Rehn M, Pirskanen A, Rautanen A, Zucchelli M, Gullsten H,
Leino M, Alenius H, Petays T, Haahtela T, Laitinen A, Laprise C, Hudson TJ, Laitinen LA, Kere J: Characterization of a common susceptibility locus for asthma-related traits. Science 2004,
304:300-304.
Kormann MS, Carr D, Klopp N, Illig T, Leupold W, Fritzsch C, Weiland SK, von Mutius E, Kabesch M: G-Protein-coupled receptor
polymorphisms are associated with asthma in a large German population. Am J Respir Crit Care Med 2005, 171:1358-1362.
Xu YL, Reinscheid RK, Huitron-Resendiz S, Clark SD, Wang Z, Lin
SH, Brucher FA, Zeng J, Ly NK, Henriksen SJ, de Lecea L, Civelli O:
Neuropeptide S: a neuropeptide promoting arousal and anxiolytic-like effects. Neuron 2004, 43:487-497.
Perez-Torres S, Miro X, Palacios JM, Cortes R, Puigdomenech P, Mengod G: Phosphodiesterase type 4 isozymes expression inhuman brain examined by in situ hybridization histochemistry and [3H]rolipram binding autoradiography. Comparison
with monkey and rat brain. J Chem Neuroanat 2000, 20:349-374.
Gretarsdottir S, Thorleifsson G, Reynisdottir ST, Manolescu A, Jonsdottir S, Jonsdottir T, Gudmundsdottir T, Bjarnadottir SM, Einarsson
OB, Gudjonsdottir HM, Hawkins M, Gudmundsson G, Gudmundsdottir H, Andrason H, Gudmundsdottir AS, Sigurdardottir M, Chou
TT, Nahmias J, Goss S, Sveinbjornsdottir S, Valdimarsson EM, Jakob-

http://www.biomedcentral.com/1471-2350/8/S1/S9

50.
51.
52.

sson F, Agnarsson U, Gudnason V, Thorgeirsson G, Fingerle J, Gurney
M, Gudbjartsson D, Frigge ML, Kong A, Stefansson K, Gulcher JR:
The gene encoding phosphodiesterase 4D confers risk ofischemic stroke. Nat Genet 2003, 35:131-138.
Worrall BB, Mychaleckyj JC: PDE4D and stroke: a real advanceor a case of the Emperor's new clothes? Stroke 2006,
37:1955-1957.
Nehlig A, Daval JL, Debry G: Caffeine and the central nervous
system: mechanisms of action, biochemical, metabolic andpsychostimulant effects. Brain Res Brain Res Rev 1992, 17:139-170.
Lelkes Z, Alfoldi P, Erdos A, Benedek G: Rolipram, an antidepressant that increases the availability of cAMP, transiently
enhances wakefulness in rats. Pharmacol Biochem Behav 1998,
60:835-839.

Publish with Bio Med Central and every
scientist can read your work free of charge"BioMed Central will be the most significant development for
disseminating the results of biomedical researc h in our lifetime."
Sir Paul Nurse, Cancer Research UKYour research papers will be:available free of charge to the entire biomedical community
peer reviewed and published immediately upon acceptancecited in PubMed and archived on PubMed Central
yours — you keep the copyright

BioMedcentral

Submit your manuscript here:
http://www.biomedcentral.com/info/publishing_adv.asp

Page 8 of 8
(page number not for citation purposes)