Hypermethylation of Proopiomelanocortin and Period 2 Genes in Blood Are Associated with Greater Subjective and Behavioral Motivation for Alcohol in Humans.

BACKGROUND: Epigenetic modifications of a gene have been shown to play a role in maintaining a long-lasting change in gene expression. We hypothesize that alcohol's modulating effect on DNA methylation on certain genes in blood is evident in binge and heavy alcohol drinkers and is associated with alcohol motivation. METHODS: Methylation-specific polymerase chain reaction (PCR) assays were used to measure changes in gene methylation of period 2 (PER2) and proopiomelanocortin (POMC) genes in peripheral blood samples collected from nonsmoking moderate, nonbinging, binge, and heavy social drinkers who participated in a 3-day behavioral alcohol motivation experiment of imagery exposure to either stress, neutral, or alcohol-related cues, 1 per day, presented on consecutive days in counterbalanced order. Following imagery exposure on each day, subjects were exposed to discrete alcoholic beer cues followed by an alcohol taste test (ATT) to assess behavioral motivation. Quantitative real-time PCR was used to measure gene expression of PER2 and POMC gene levels in blood samples across samples. RESULTS: In the sample of moderate, binge, and heavy drinkers, we found increased methylation of the PER2 and POMC DNA, reduced expression of these genes in the blood samples of the binge and heavy drinkers relative to the moderate, nonbinge drinkers. Increased PER2 and POMC DNA methylation was also significantly predictive of both increased levels of subjective alcohol craving immediately following imagery (p < 0.0001), and with presentation of the alcohol (2 beers) (p < 0.0001) prior to the ATT, as well as with alcohol amount consumed during the ATT (p < 0.003). CONCLUSIONS: These data establish significant association between binge or heavy levels of alcohol drinking and elevated levels of methylation and reduced levels of expression of POMC and PER2 genes. Furthermore, elevated methylation of POMC and PER2 genes is associated with greater subjective and behavioral motivation for alcohol.

Glucocorticoids entrain molecular clock components in human peripheral cells.

In humans, shift work induces a desynchronization between the circadian system and the outside world, which contributes to shift work-associated medical disorders. Using a simulated night shift experiment, we previously showed that 3 d of bright light at night fully synchronize the central clock to the inverted sleep schedule, whereas the peripheral clocks located in peripheral blood mononuclear cells (PBMCs) took longer to reset. This underlines the need for testing the effects of synchronizers on both the central and peripheral clocks. Glucocorticoids display circadian rhythms controlled by the central clock and are thought to act as synchronizers of rodent peripheral clocks. In the present study, we tested whether the human central and peripheral clocks were sensitive to exogenous glucocorticoids (Cortef) administered in the late afternoon. We showed that 20 mg Cortef taken orally acutely increased PER1 expression in PBMC peripheral clocks. After 6 d of Cortef administration, the phases of central markers were not affected, whereas those of PER2-3 and BMAL1 expression in PBMCs were shifted by ∼ 9.5-11.5 h. These results demonstrate, for the first time, that human peripheral clocks are entrained by glucocorticoids. Importantly, they suggest innovative interventions for shift workers and jet-lag travelers, combining synchronizing agents for the central and peripheral clocks.

Circadian clock gene expression is impaired in gestational diabetes mellitus.

Dysfunction of the circadian clock genes is involved in the development of obesity and type 2 diabetes (T2D). Since gestational diabetes mellitus (GDM) and T2D share common genetic and phenotypic features, in the present study, we investigated the status of the circadian clock in a cohort of 40 Greek pregnant women with GDM, four with T2D and 20 normal controls. Peripheral blood mRNA transcript levels of 10 clock genes (CLOCK1, BMAL1, PER1, PER2, PER3, PPARΑ, PPARD, PPARG, CRY1 and CRY2) were determined by real-time quantitative PCR. GDM patients expressed significantly lower transcript levels of BMAL1, PER3, PPARD and CRY2 compared to control women (p < 0.05). No significant difference was documented between GDM women maintained either under insulin treatment or diet. A positive correlation was found between the expression of BMAL1 versus CRY2 (r = 0.45, p = 0.003) and BMAL1 versus PPARD (r = 0.43, p = 0.004). Further investigation on the functional relevance of these clock genes, disclosed that expression of PER3 correlated negatively with HbA1C levels (r = -0.36, p = 0.022). These data document for the first time that the expression of BMAL1, PER3, PPARD and CRY2 genes is altered in GDM compared to normal pregnant women and support the notion that deranged expression of clock genes may play a pathogenic role in GDM.

The impact of sleep deprivation and nighttime light exposure on clock gene expression in humans.

AIM: To examine the effect of acute sleep deprivation under light conditions on the expression of two key clock genes, hPer2 and hBmal1, in peripheral blood mononuclear cells (PBMC) and on plasma melatonin and cortisol levels. METHODS: Blood samples were drawn from 6 healthy individuals at 4-hour intervals for three consecutive nights, including a night of total sleep deprivation (second night). The study was conducted in April-June 2006 at the University Medical Centre Ljubljana. RESULTS: We found a significant diurnal variation in hPer2 and hBmal1 expression levels under baseline (P<0.001, F=19.7, df=30 for hPer2 and P<0.001, F=17.6, df=30 for hBmal1) and sleep-deprived conditions (P<0.001, F=9.2, df=30 for hPer2 and P<0.001, F=13.2, df=30 for hBmal1). Statistical analysis with the single cosinor method revealed circadian variation of hPer2 under baseline and of hBmal1 under baseline and sleep-deprived conditions. The peak expression of hPer2 was at 13:55 ± 1:15 hours under baseline conditions and of hBmal1 at 16:08 ± 1:18 hours under baseline and at 17:13 ± 1:35 hours under sleep-deprived conditions. Individual cosinor analysis of hPer2 revealed a loss of circadian rhythm in 3 participants and a phase shift in 2 participants under sleep-deprived conditions. The plasma melatonin and cortisol rhythms confirmed a conventional alignment of the central circadian pacemaker to the habitual sleep/wake schedule. CONCLUSION: Our results suggest that 40-hour acute sleep deprivation under light conditions may affect the expression of hPer2 in PBMCs..

Expression of HO1 and PER2 can predict the incidence of delirium in trauma patients with concomitant brain injury.

Intensive care unit (ICU)-acquired delirium is associated with adverse outcome in trauma patients with concomitant traumatic brain injury (TBI), but diagnosis remains challenging. Quantifying circadian disruption by analyzing expression of the circadian gene period circadian regulator 2 (PER2) and heme oxygenase 1 (HO1), which determines heme turnover, may prove to be potential diagnostic tools. Expression of PER2 and HO1 was quantified using qPCR from blood samples 1 day and 7 days after trauma. Association analysis was performed comparing mRNA expression levels with parameters of trauma (ISS-injury severity score), delirium, acute kidney injury (AKI) and length of ICU stay. 48 polytraumatized patients were included (equal distribution of TBI versus non-TBI) corrected for ISS, age and gender using a matched pairs approach. Expression levels of PER2 and HO1 were independent of age (PER2: P = 0.935; HO1: P = 0.988), while expression levels were significantly correlated with trauma severity (PER2: P = 0.009; HO1: P < 0.001) and longer ICU length of stay (PER2: P = 0.018; HO1: P < 0.001). High expression levels increased the odds of delirium occurrence (PER2: OR = 4.32 [1.14-13.87]; HO1: OR = 4.50 [1.23-14.42]). Patients with TBI showed a trend towards elevated PER2 (OR = 3.00 [0.84-9.33], P = 0.125), but not towards delirium occurrence (P = 0.556). TBI patients were less likely to develop AKI compared to non-TBI (P = 0.022). Expression levels of PER2 and HO1 correlate with the incidence of delirium in an age-independent manner and may potentially improve diagnostic algorithms when used as delirium biomarkers.Trial registration: German Clinical Trials Register (Trial-ID DRKS00008981; Universal Trial Number U1111-1172-6077; Jan. 18, 2018).

Expression of clock genes Per1 and Bmal1 in total leukocytes in health and Parkinson's disease.

BACKGROUND: There is a growing number of clinical studies that revealed a variety of behavioral and physiological desynchronies in patients with Parkinson's disease (PD). However, these desynchronies have not been defined at the molecular level. METHODS: Using real-time RT-PCR assay, we analyzed the expression profiles of two principle clock genes, PER1 and BMAL1, in total leukocytes for 12 h during the evening, overnight and morning in subjects with PD and age/sex-matched healthy controls. RESULTS: A difference in the expression pattern of BMAL1 but not PER1 was apparent during the dark span, where the relative abundance of BMAL1 was significantly lower in PD patients versus control subjects at 21:00, 00:00 and 06:00 h. Furthermore, expression levels of BMAL1 in PD patients correlated with their United Parkinson's Disease Rating Scale score at 06:00, 09:00 h, and with Pittsburgh Sleep Quality Index score at 06:00 h. CONCLUSION: These results suggest that a peripheral molecular clock, as reflected in the dampened expression of the clock genes BMAL1 in total leukocytes, is altered in PD patients. In addition, the relative BMAL1 levels correlate positively with PD severity, which could provide a molecular basis to help monitor disease progression and response to investigational drugs.

Effect of melatonin administration on the PER1 and BMAL1 clock genes in patients with Parkinson's disease.

Sleep disorders are a widespread condition in patients with Parkinson's disease (PD), which has been linked to a deregulation of the circadian cycle and therefore of the clock genes. The aim of this study was to evaluate the effect of melatonin (MEL) on the PER1 and BMAL1 clock genes in patients with PD. A double-blind, cross-over, placebo-controlled randomized clinical trial pilot study was conducted in 26 patients with stage 1-3 PD according to the Hoehn & Yahr scale, who received either 25 mg of MEL or a placebo at noon and 30 min before bedtime for three months. The relative expression of the PER1 and BMAL1 genes was measured, as well as the presence of daytime, nocturnal, and global sleepiness, and the progression of PD. The levels of the PER1 and BMAL1 genes at baseline were 0.9 (0.1-3) vs. 0.56 (0.1-2.5), respectively; while after the intervention with MEL or placebo the BMAL1 levels increased to 2.5 (0-3.70) vs. 2.2 (0.10-3.30), respectively (d = 0.387). Fifty percent (50 %) of patients had daytime sleepiness and sixty-five percent (65 %) had abnormal nighttime sleepiness, yet neither group showed changes after the intervention. Patients with PD exhibited an alteration in the levels of the clock genes: MEL increased the levels of BMAL1, but the PER1 levels remained unchanged.

Regulation of Circadian Clock Genes on Sleep Disorders in Traumatic Brain Injury Patients.

BACKGROUND: In patients with traumatic brain injury (TBI), whether sleep disorder is associated with disturbances in molecular rhythmicity is unclear. This study aimed to investigate the relationship between abnormal sleep and regulation by circadian rhythms in patients with TBI. METHODS: We sampled buccal cells and human blood samples from patients with TBI diagnosed with sleep disorders and those with normal sleep and investigated differences in the expression levels of Clock, Per2, and Bmal1 between the 2 groups. RESULTS: The expression peaks of Clock, Per2, and Bmal1 were at 12:00. There was a statistically significant difference between the sleep disorder group and the normal sleep group in the level of Clock mRNA expression (P = 0.0003 in oral mucosa and P < 0.0001 in mononuclear cells). There was no significant between-group difference in Bmal1 mRNA expression level (P = 0.1187 in oral mucosa and P = 0.2094 in mononuclear cells). There were significant between-group differences in Per2 mRNA expression levels at 12:00 (P = 0.0102 in oral mucosa and P = 0.0006 in mononuclear cells) and 18:00 (P = 0.0004 in oral mucosa and P = 0.0015 in mononuclear cells) but no significant difference at 24:00 (P = 0.7838 in oral mucosa and P = 0.2808 in mononuclear cells). CONCLUSIONS: Abnormal expression levels of Per2, Clock, and Bmal1 were detected in patients with TBI-related sleep disorders. These novel findings demonstrate disturbances in the molecular clock in TBI patients and have important implications for our understanding of the aberrant rhythms reported in this disease.

Circadian Rhythm Disruption and Sepsis in Severe Trauma Patients.

BACKGROUND: Circadian rhythms are important regulators of immune functions. Admission to an intensive care unit may impact molecular clock activity and host response. Our objective was to assess and compare the immune circadian rhythms in trauma patients who develop and in those who do not develop sepsis. METHODS: Blood samples were collected from severe trauma patients within 4 days after admission, with collections taking place every 4 h over a 24-h period. Cortisol and cytokines were measured with immunoassays. Whole-blood expression of 3 clock genes (Bmal1, Per2, and Per3) was studied by reverse transcription quantitative polymerase chain reaction. Neutrophils, monocytes, and lymphocytes were analyzed by flow cytometry. Patients with and without sepsis were compared with the cosinor mixed model to estimate mesors, amplitudes, and acrophases. RESULTS: Thirty-eight patients were enrolled in the study, and 13 developed at least 1 septic episode. The septic patients had higher levels of cortisol than the nonseptic patients (mesor at 489 nmol/L vs. 405 nmol/L, P < 0.05) and delayed acrophases (22 h vs. 15 h, P < 0.05). They also had lower lymphocyte counts (mesor at 785 vs. 1,012 cells/µL, P < 0.05), higher neutrophil counts (mesor at 7,648 vs. 7,001 cells/µL, P < 0.05), and monocyte counts (mesor at 579 vs. 473 cells/µL, P < 0.05) than the nonseptic patients. Although no amplitude difference was identified, the acrophases were significantly different between the 2 groups for lymphocytes, interleukin 10 and tumor necrosis factor. CONCLUSION: We demonstrated that all trauma patients had impaired circadian rhythms of cortisol, cytokines, leukocytes, and clock genes. Early circadian disruption was associated with the occurrence of sepsis and might be a marker of sepsis severity.

Diurnal neurobiological alterations after exposure to clozapine in first-episode schizophrenia patients.

BACKGROUND: Irregular circadian rhythm and some of its most characteristic symptoms are frequently observed in patients with schizophrenia. However, changes in the expression of clock genes or neuropeptides that are related to the regulation of circadian rhythm may influence the susceptibility to recurrence after antipsychotic treatment in schizophrenia, but this possibility has not been investigated. METHODS: Blood samples were collected from 15 healthy male controls and 13 male schizophrenia patients at 4h intervals for 24h before and after treatment with clozapine for 8 weeks. The outcome measures included the relative expression of clock gene mRNA PERIOD1 (PER1), PERIOD2 (PER2), PERIOD3 (PER3) and the levels of plasma cortisol, orexin, and insulin. RESULTS: Compared with healthy controls, schizophrenia patients presented disruptions in diurnal rhythms of the expression of PER1, PER3, and NPAS2 and the release of orexin, accompanied by a delayed phase in the expression of PER2, decreases in PER3 and NPAS2 expression, and an increase in cortisol levels at baseline. Several of these disruptions (i.e., in PER1 and PER3 expression) persisted after 8 weeks of clozapine treatment, similar to the decreases in the 24-h expression of PER3 and NPAS2. Clozapine treatment for 8 weeks significantly decreased the 24-h levels of PER2 and increased the 24-h levels of insulin. CONCLUSION: These persistent neurobiological changes that occur after 8 weeks of clozapine treatment may contribute to the vulnerability to recurrence and efficacy of long-term maintenance treatment in schizophrenia.

miR-92a Corrects CD34+ Cell Dysfunction in Diabetes by Modulating Core Circadian Genes Involved in Progenitor Differentiation.

Autologous CD34(+) cells are widely used for vascular repair; however, in individuals with diabetes and microvascular disease these cells are dysfunctional. In this study, we examine expression of the clock genes Clock, Bmal, Per1, Per2, Cry1, and Cry2 in CD34(+) cells of diabetic and nondiabetic origin and determine the small encoding RNA (miRNA) profile of these cells. The degree of diabetic retinopathy (DR) was assessed. As CD34(+) cells acquired mature endothelial markers, they exhibit robust oscillations of clock genes. siRNA treatment of CD34(+) cells revealed Per2 as the only clock gene necessary to maintain the undifferentiated state of CD34(+) cells. Twenty-five miRNAs targeting clock genes were identified. Three of the miRNAs (miR-18b, miR-16, and miR-34c) were found only in diabetic progenitors. The expression of the Per2-regulatory miRNA, miR-92a, was markedly reduced in CD34(+) cells from individuals with DR compared with control subjects and patients with diabetes with no DR. Restoration of miR-92a levels in CD34(+) cells from patients with diabetes with DR reduced the inflammatory phenotype of these cells and the diabetes-induced propensity toward myeloid differentiation. Our studies suggest that restoring levels of miR-92a could enhance the usefulness of CD34(+) cells in autologous cell therapy.

Per3 length polymorphism in patients with type 2 diabetes mellitus.

BACKGROUND: A number of observations support the involvement of circadian clock genes in the regulation of metabolic processes. One of these circadian genes, Per3, exhibits a variable number tandem repeat length polymorphism, consisting of two alleles, namely four and five repeat alleles, in its exon 18. The objective of this study was to examine the existence of Per3 variants in patients with type 2 diabetes mellitus (T2DM) as compared to a non T2DM control group. METHODS: Intravenous blood samples were collected to obtain white blood cells from 302 T2DM patients and 330 non-diabetic, age- and sex-matched, individuals. Per3 genotyping was performed on DNA by polymerase chain reaction. RESULTS: Frequency of five repeat allele was higher, and that of four repeat allele lower, in T2DM patients as compared to non-diabetic controls (χ2=6.977, p=0.0082) CONCLUSIONS: The results indicate an association of Per3 five repeat allele with T2DM occurrence and suggest that individuals with five repeat allele may be at a greater risk for T2DM as compared to those carrying the four repeat allele.

Early sex-specific modulation of the molecular clock in trauma.

BACKGROUND: Immune system biology and most physiologic functions are tightly linked to circadian rhythms. Time of day-dependent variations in many biologic parameters also play a fundamental role in the disease process. We previously showed that the genes encoding the peripheral molecular clock were modulated in a sex-dependent manner in Q fever. METHODS: Here, we examined severe trauma patients at admission to the intensive care unit. Using quantitative real-time polymerase chain reaction, the whole-blood expression of the molecular clock components ARNTL, CLOCK, and PER2 was assessed in male and female trauma patients. Healthy volunteers of both sexes were used as controls. RESULTS: We observed a significant overexpression of both ARNTL and CLOCK in male trauma patients. CONCLUSION: We report, for the first time, the sex-related modulation of the molecular clock genes in the blood following severe trauma. These results emphasize the role of circadian rhythms in the immune response in trauma patients. LEVEL OF EVIDENCE: Epidemiologic study, level IV.

PER1 and CLOCK: potential circulating biomarkers for head and neck squamous cell carcinoma.

BACKGROUND: Circadian clock regulates daily rhythms in various physiologic processes and deregulated circadian clock are linked to cancers. We have previously demonstrated the association between altered circadian clock genes (CCGs) and head and neck squamous cell carcinoma (HNSCC). The purpose of this study was to investigate whether the CCGs were also altered in peripheral blood (PB) of patients with HNSCC. METHODS: The 9 CCGs expression profiles of PB leukocytes from 94 patients with HNSCC and 56 healthy individuals were investigated using real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and immunocytochemistry. RESULTS: All the 9 CCGs were significantly downregulated in the PB of preoperative patients (p < .0001). Recovery of PER1 and CLOCK expression was observed in postoperative patients with good prognosis but not in patients that died within 1 year after surgery. CONCLUSION: CCGs were also altered in PB leukocytes of patients with HNSCC and PER1 and CLOCK are potential circulating prognostic markers for HNSCC.

The clock gene Per2 is required for normal platelet formation and function.

INTRODUCTION: Apoptotic cell death is a highly regulated genetic program, which has been observed in mature megakaryocytes fragmenting into platelets. The clock gene Per2, a key component of core clock oscillator, was involved in affecting both cell cycle control and apoptosis. Thus, loss of Per2 function may be considered potential influence of platelet formation and function. METHODS: Per2-null mice and C57BL/6 mice were used in the study. Bleeding time, platelet count, megakaryocyte count, megakaryocyte ploidy, megakaryocyte apoptosis, rate of proplatelet formation, clot retraction, platelet aggregation and secretion were performed to evaluate thrombopoiesis and hemostasis. Quantitative RT-PCR was employed to analyze genes expression in liver, bone marrow and enriched megakaryocytes. RESULTS: The Per2-null mice had nearly 50% platelet counts in peripheral blood. Per2-null platelets were compromised in their ability to aggregate and secretion, consistent with a marked reduction in the number of dense and a-granules. Megakaryocytes from Per2-null mice showed no significant variation in number but increased in ploidy. Ultrastructural examination of Per2-null megakaryocytes revealed many vacuoles in demarcation membranes and reduction in platelet granules. Megakaryocytes from Per2-null bone marrow decreased the rate of proplatelet formation and impaired apoptosis. Per2-null mice showed increased both in Tpo in livers and its receptors C-mpl in bone marrow, and the megakaryocytes from these mice decreased P53 expression, consequently increased Bcl-xl and Bcl-2 level. CONCLUSIONS: The clock gene Per2 modulating the apoptosis of megakaryocytes was required for platelet formation and function.

Influence of age on clock gene expression in peripheral blood cells of healthy women.

Recent studies have demonstrated a close relationship between circadian clock function and the development of obesity and various age-related diseases. In this study, we investigated whether messenger RNA (mRNA) levels of clock genes are associated with age, body mass index, blood pressures, fasting plasma glucose, or shift work. Peripheral blood cells were obtained from 70 healthy women, including 25 shift workers, at approximately 9:00 AM. Transcript levels of clock genes (CLOCK, BMAL1, PER1, and PER3) were determined by real-time quantitative polymerase chain reaction. Stepwise multiple regression analysis demonstrated that BMAL1 mRNA levels were correlated only with age (beta = -.50, p < .001). In contrast, PER3 levels were correlated with fasting plasma glucose (beta = -.29, p < .05) and shift work (beta = .31, p < .05). These results suggest that increased age, glucose intolerance, and irregular hours independently affect the intracellular clock in humans.