Professor Malcolm von Schantz

Our own species has a diurnal activity pattern and an average circadian period of 24.2 hours. Exact determination of circadian period requires expensive and intrusive protocols, and investigators are therefore using chronotype questionnaires as a proxy quantitative measure. Both measures show a normal distribution suggestive of a polygenic trait. The genetic components of the 24-hour feedback loop that generates circadian rhythms within our cells have been mapped in detail, identifying a number of candidate genes which have been investigated for genetic polymorphisms relating to the phenotypic variance. Key in this mechanism is the inhibitory complex containing period and cryptochrome proteins and interacting protein kinases and ubiquitin ligases, and the stability of this complex is recognized as the major determinant of circadian periodicity. The identification of the causative mutations in familial circadian rhythms sleep disorders has shed additional light into this mechanism. Mutations in the negative feedback protein-encoding genes PER2 and CRY2 as well as the CSNK1D gene encoding casein kinase I delta have been shown to cause advanced sleep phase disorder, and a mutation in the CRY1 gene delayed sleep phase disorder. The candidate gene approach has also yielded a number of genetic associations with chronotype as determined by questionnaires. More recently, genome-wide association studies (GWAS) of chronotype have both confirmed associations with the candidate clock gene PER2 and identified a serious of novel genes associated with variability in circadian rhythmicity, which have yet to be explored. Whilst considerable progress has thus been made with mapping the phenotypic diversity in human circadian rhythms and the genomic variability that causes it, studies to date have been mostly focused on individuals of European descent, and there is a strong need for research on other populations.

The well-established negative health outcomes of sleep deprivation, and the suggestion that
availability of electricity may enable later bed times without compensating sleep extension in the
morning, have stimulated interest in studying communities whose sleep pattern may resemble a preindustrial
state. Here, we describe sleep and activity in two neighbouring communities, one urban
(Milange) and one rural (Tengua), in a region of Mozambique where urbanisation is an ongoing process.
The two communities differ in the amount and timing of daily activity and of light exposure, with later
bedtimes (H1 h) associated with more evening and less daytime light exposure seen in the town of
Milange. In contrast to previous reports comparing communities with and without electricity, sleep
duration did not differ between Milange (7.28 h) and Tengua (7.23 h). Notably, calculated sleep quality
was significantly poorer in rural Tengua than in Milange, and poor sleep quality was associated with
a number of attributes more characteristic of rural areas, including more intense physical labour and
less comfortable sleeping arrangements. Thus, whilst our data support the hypothesis that access to
electricity delays sleep timing, the higher sleep quality in the urban population also suggests that some
aspects of industrialisation are beneficial to sleep.

Circadian rhythms organize many aspects of cell biology and physiology to a daily temporal program that depends on clock gene expression cycles in most mammalian cell types. However, circadian rhythms are also observed in isolated mammalian red blood cells (RBCs), which lack nuclei, suggesting the existence of post-translational cellular clock mechanisms in these cells. By using electrophysiological and pharmacological approaches, we show that human RBCs display circadian regulation of membrane conductance and cytoplasmic conductivity that depends on the cycling of cytoplasmic K+ levels. Using pharmacological intervention and ion replacement, we show that inhibition of K+ transport abolishes RBC electrophysiological rhythms. Our results suggest that in the absence of conventional transcription cycles, RBCs maintain a circadian rhythm in membrane electrophysiology through dynamic regulation of K+ transport.

Later chronotype (i.e. evening preference) and later timing of sleep have been associated with greater
morbidity, including higher rates of metabolic dysfunction and cardiovascular disease. However, no one has
examined whether chronotype is associated with mortality risk to date. Our objective was to test the
hypothesis that being an evening type is associated with increased mortality in a large cohort study, the UK
Biobank. Our analysis included 433,268 adults aged 38-73 at the time of enrolment and an average 6.5-
year follow-up. The primary exposure was chronotype, as assessed through a single self-reported question
defining participants as definite morning types, moderate morning types, moderate evening types, or
definite evening types. The primary outcomes were all-cause mortality and mortality due to cardiovascular
disease (CVD). Prevalent disease was also compared among the chronotype groups. Analyses were
adjusted for age, sex, ethnicity, smoking, body mass index, sleep duration, socioeconomic status and
comorbidities. Greater eveningness, particularly being a definite evening type, was significantly associated
with a higher prevalence of all comorbidities. Comparing definite evening type to definite morning type, the
associations were strongest for psychological disorders (OR 1.94, 95% CI 1.86 to 2.02, p=<.001 followed=""> by diabetes (OR 1.30, 95% CI 1.24 to 1.36, p=<.001 neurological="" disorders="" ci="" to=""> 1.30, p=<.001 gastrointestinal="" abdominal="" disorders="" ci="" to="" p="<.001)," and=""> respiratory disorders (OR 1.22, 95% CI 1.18 to 1.26, p=<.001 the="" total="" number="" of="" deaths="" was="" out=""> of which 2,127 were due to CVD. Greater eveningness, based on chronotype as an ordinal variable, was
associated with a small increased risk of all-cause mortality (HR 1.02, 95% CI 1.004 to 1.05, p=.017) and
CVD mortality (HR 1.04, 95% CI 1.00 to 1.09, p=.06). Compared to definite morning types, definite evening
types had significantly increased risk of all-cause mortality (HR 1.10, 95% CI 1.02 to 1.18, p=.012). This
first report of increased mortality in evening types is consistent with previous reports of increased levels of
cardiometabolic risk factors in this group. Mortality risk in evening types may be due to behavioural,
psychological, and physiological risk factors, many of which may be attributable to chronic misalignment
between internal physiological timing and externally imposed timing of work and social activities. These
findings suggest the need for researching possible interventions aimed at either modifying circadian
rhythms in individuals or at allowing evening types greater working hour flexibility.

Circadian rhythms and sleep are two separate but intimately related processes. Circadian rhythms are generated through the precisely controlled, cyclic expression of a number of genes designated clock genes. Genetic variability in these genes has been associated with a number of phenotypic differences in circadian, but also in sleep parameters both in mouse models and in humans. Diurnal preferences, as determined by the self-reported Horne-Östberg questionnaire, has been associated with polymorphisms in the human genes CLOCK, PER1, PER2, and PER3. Circadian rhythms sleep disorders have also been associated with mutations and polymorphisms in clock genes, with the advanced type cosegrating in an autosomal dominant inheritance pattern with mutations in the genes PER2 and CSNK1D, and the delayed type associating without discernible Mendelian inheritance with polymorphisms in CLOCK and PER3. Several mouse models of clock gene null alleles have been demonstrated to have affected sleep homeostasis. Recent findings have shown that the variable number tandem polymorphism in PER3, previously linked to diurnal preference, has profound effects on sleep homeostasis and cognitive performance following sleep loss, confirming the close association between the processes of circadian rhythms and sleep on the genetic level.

In recent years, strong evidence has emerged suggesting that insufficient duration, quality, and/or timing of sleep are associated with cardiovascular disease (CVD), and various mechanisms for this association have been proposed. Such associations may be related to endophenotypic features of the sleep homeostat and the circadian oscillator, or may be state-like effects of the environment. Here, we review recent literature on sleep, circadian rhythms and CVD with a specific emphasis on differences between racial/ethnic groups. We discuss the reported differences, mainly between individuals of European and African descent, in parameters related to sleep (architecture, duration, quality) and circadian rhythms (period length and phase shifting). We further review racial/ethnic differences in cardiovascular disease and its risk factors, and develop the hypothesis that racial/ethnic health disparities may, to a greater or smaller degree, relate to differences in parameters related to sleep and circadian rhythms. When humans left Africa some 100,000 years ago, some genetic differences between different races/ethnicities were acquired. These genetic differences have been proposed as a possible predictor of CVD disparities, but concomitant differences in culture and lifestyle between different groups may equally explain CVD disparities. We discuss the evidence for genetic and environmental causes of these differences in sleep and circadian rhythms, and their usefulness as health intervention targets.

In spite of suspected circadian differences between different ancestral groups, most human studies have used individuals of European descent. This also applies to three recent genome-wide association studies (GWAS), which pinpointed a number of chronotype loci. We investigated the distribution of these hits in different 1000 Genomes populations. We found six out of the 41 alleles previously identified by GWAS in European participants (in the genes RGS16, PER2, AK5, and between the genes APH1A and CA14) to be absent from some non-European population groups. This highlights the need for ancestral diversity in circadian research, and may reflect differences affecting the phenotype of individuals of East Asian ancestry.

As part of the Health and Aging in Africa: A Longitudinal Study of an INDEPTH Community in
South Africa (HAALSI), we investigated sleep habits and their interactions with HIV or noncommunicable
diseases (NCDs) in 5059 participants (median age: 61, interquartile range:
52?71, 54% females). Self-reported sleep duration was 8.2±1.6h, and bed and rise times
were 20:48±1:15 and 05:31±1:05 respectively. Ratings of insufficient sleep were associated
with older age, lack of formal education, unemployment, and obesity (p restless sleep were associated with being older, female, having more education, being
unemployed, and single. Hypertension was associated with shorter self-reported sleep
duration, poor sleep quality, restless sleep, and periods of stopping breathing during the
night (p nocturnal awakenings than those on ART (p=0.029) and HIV negative individuals (p=0.024),
suggesting a negative net effect of untreated infection, but not of ART, on sleep quality. In
this cohort, shorter, poor-quality sleep was associated with hypertension, but average self-reported
sleep duration was longer than reported in other regions globally. It remains to be
determined whether this is particular to this cohort, South Africa in general, or low- to
middle-income countries undergoing transition.

Aim: To test the association between cardiometabolic risk factors and
subjective sleep quality assessed by the Pittsburgh sleep quality index
(PSQI), independent of obstructive sleep apnea (OSA) and sleep duration.

Methods: 573 participants from the Baependi Heart Study, a rural cohort
from Brazil that completed sleep questionnaires and underwent polygraphy
for OSA evaluation. Multivariable linear regression analysis tested the
association between cardiovascular risk factors (outcome variables) and
sleep quality measured by PSQI, adjusting for OSA and other potential
confounders (age, sex, race, salary/wage, education, marital status,
alcohol intake, obesity, smoking, hypertension, and sleep duration).

Results: The sample mean age was of 43±16y, 66% were female, and mean
body mass index (BMI) was 26±5 kg/m2. Only 20% were classified as obese
(BMI e30). Fifty percent of participants reported poor sleep quality as
defined by a PSQI score e5. A high PSQI score was significantly
associated with higher very-low density lipoprotein (VLDL) cholesterol
levels (beta=0.392, p=0.012) and higher triglyceride levels (beta=0.017,
p=0.006), even after adjustments, including the apnea-hypopnea index.
Further adjustments accounting for marital status, alcohol intake, and
medication use did not change these findings. No significant association
was observed between PSQI scores and glucose or blood pressure. According
to PSQI components, sleep disturbances (beta=1.976, p=0.027), sleep
medication use (beta=1.121, p=0.019), and daytime dysfunction
(beta=1.290, p=0.024) were significantly associated with higher VLDL
serum levels. Only the daytime dysfunction domain of the PSQI components
was significantly associated with higher triglyceride levels (beta=0.066,
p=0.004).

Conclusion: Poorer lipid profile was independently associated with poor
sleep quality, assessed by the PSQI questionnaire, regardless of a normal
sleep duration and accounting for OSA and socio-economic status.

Cardiometabolic risk factors influence white matter hyperintensity (WMH) development: in metabolic syndrome (MetS), higher WMH load is often reported but the relationships between specific cardiometabolic variables, WMH load and cognitive performance are uncertain. We investigated these in a Brazilian sample (aged 50-85) with (N=61) and without (N=103) MetS. Stepwise regression models identified effects of cardiometabolic and demographic variables on WMH load (from FLAIR MRI) and verbal recall performance. WMH volume was greater in MetS, but verbal recall performance was not impaired. Age showed the strongest relationship with WMH load. Across all participants, systolic blood pressure (SBP) and fasting blood glucose were also contributors, and WMH volume was negatively associated with verbal recall performance. In non-MetS, higher HbA1c, SBP, and number of MetS components were linked to poorer recall performance while higher triglyceride levels appeared to be protective. In MetS only, these relationships were absent but education exerted a strongly protective effect on recall performance. Thus, results support MetS as a construct: the clustering of cardiometabolic variables in MetS alters their individual relationships with cognition; instead, MetS is characterised by a greater reliance on cognitive reserve mechanisms. In non-MetS, strategies to control HbA1c and SBP should be prioritised as these have the largest impact on cognition.

Temperature compensation and period determination by casein kinase 1 (CK1) are conserved features of eukaryotic circadian rhythms, whereas the clock gene transcription factors that facilitate daily gene expression rhythms differ between phylogenetic kingdoms. Human red blood cells (RBCs) exhibit temperature compensated circadian rhythms which, since RBCs lack nuclei, must occur in the absence of a circadian transcription-translation feedback loop. We tested whether period determination and temperature compensation are dependent on casein kinases in RBCs. As with nucleated cell types, broad spectrum kinase inhibition with staurosporine lengthened the period of the RBC clock at 37°C, with more specific inhibition of CK1 and CK2 also eliciting robust changes in circadian period. Strikingly, inhibition of CK1 abolished temperature compensation and increased the Q10 for the period of oscillation in RBCs, similar to observations in nucleated cells. This indicates that CK1 activity is essential for circadian rhythms irrespective of the presence or absence of clock gene expression cycles.

Individual variability in word generation is a product of genetic and environmental influences. The genetic effects on semantic verbal fluency were estimated in 1,735 participants from the Brazilian Baependi Heart Study. The numbers of exemplars produced in 60 s were broken down into time quartiles because of the involvement of different cognitive processes ? predominantly automatic at the beginning, controlled/executive at the end. Heritability in the unadjusted model for the 60-s measure was 0.32. The best-fit model contained age, sex, years of schooling, and time of day as covariates, giving a heritability of 0.21. Schooling had the highest moderating effect. The highest heritability (0.17) was observed in the first quartile, decreasing to 0.09, 0.12, and 0.0003 in the following ones. Heritability for average production starting point (intercept) was 0.18, indicating genetic influences for automatic cognitive processes. Production decay (slope), indicative of controlled processes, was not significant. The genetic influence on different quartiles of the semantic verbal fluency test could potentially be exploited in clinical practice and genome-wide association studies.

Temperature compensation and period determination by casein kinase 1 (CK1) are conserved features of eukaryotic circadian rhythms, whereas the clock gene transcription factors that facilitate daily gene expression rhythms differ between phylogenetic kingdoms. Human red blood cells (RBCs) exhibit temperature-compensated circadian rhythms, which, because RBCs lack nuclei, must occur in the absence of a circadian transcription-translation feedback loop. We tested whether period determination and temperature compensation are dependent on CKs in RBCs. As with nucleated cell types, broad-spectrum kinase inhibition with staurosporine lengthened the period of the RBC clock at 37°C, with more specific inhibition of CK1 and CK2 also eliciting robust changes in circadian period. Strikingly, inhibition of CK1 abolished temperature compensation and increased the Q10 for the period of oscillation in RBCs, similar to observations in nucleated cells. This indicates that CK1 activity is essential for circadian rhythms irrespective of the presence or absence of clock gene expression cycles.

Studying communities at different stages of urbanisation and industrialisation can teach us how timing and intensity of light affects the circadian clock under real-life
conditions. We have previously described a strong tendency towards morningness in the Baependi Heart Study, located in a small rural town in Brazil. Here, we tested the
hypothesis that this morningness tendency is associated with early circadian phase based on objective measurements (as determined by dim light melatonin onset,
DLMO, and activity) and light exposure. We also analysed how well the previously collected chronotype questionnaire data was able to predict these DLMO values. The average DLMO observed in 73 participants (40 female) was 20:03±01:21, SD, with an earlier average onset in men (19:38±01:16) than in women (20:24±01:21; pd0.01). However, men presented larger phase angle between DLMO and sleep onset time as
measured by actigraphy (4.11 hours vs 3.16 hours; pd0.01). Correlational analysis indicated associations between light exposure, activity rhythms, and DLMO, such that
early DLMO was observed in participants with higher exposure to light, higher activity and earlier light exposure. The strongest significant predictor of DLMO was
morningness-eveningness questionnaire (MEQ) (beta=-0.35, pd0.05), followed by age(beta=-0.47, pd0.01). Sex, light exposure, and variables derived from the Munich
Chronotype Questionnaire were not significant predictors. Our observations demonstrate that both early sleep patterns and earlier circadian phase have been retained in this small rural town in spite of availability of electrification, in contrast to metropolitan post-industrial areas.