Mario Pedrazzoli

A mutation in a case of early onset narcolepsy and a generalized absence of hypocretin peptides in human narcoleptic brains

We explored the role of hypocretins in human narcolepsy through histopathology of six narcolepsy brains and mutation screening of Hcrt, Hcrtr1 and Hcrtr2 in 74 patients of various human leukocyte antigen and family history status. One Hcrt mutation, impairing peptide trafficking and processing, was found in a single case with early onset narcolepsy. In situ hybridization of the perifornical area and peptide radioimmunoassays indicated global loss of hypocretins, without gliosis or signs of inflammation in all human cases examined. Although hypocretin loci do not contribute significantly to genetic predisposition, most cases of human narcolepsy are associated with a deficient hypocretin system.

Clock polymorphisms and circadian rhythms phenotypes in a sample of the Brazilian population

A Clock polymorphism T to C situated in the 3′ untranslated region (3′‐UTR) has been associated with human diurnal preference. At first, Clock 3111C had been reported as a marker for evening preference. However these data are controversial, and data both corroborating and denying them have been reported. This study hypothesizes that differences in Clock genotypes could be observed if extreme morning‐type subjects were compared with extreme evening‐type subjects, and the T3111C and T257G polymorphisms were studied. The possible relationship between both polymorphisms and delayed sleep phase syndrome (DSPS) was also investigated. An interesting and almost complete linkage disequilibrium between the polymorphisms T257G in the 5′ UTR region and the T3111C in the 3′ UTR region of the Clock gene is described. Almost always, a G in position 257 corresponds to a C in position 3111, and a T in position 257 corresponds to a T in position 3111. The possibility of an interaction of these two regions in the Clock messenger RNA structure that could affect gene expression was analyzed using computer software. The analyses did not reveal an interaction between those two regions, and it is unlikely that this full allele correspondence affects Clock gene expression. These results show that there is no association between either polymorphism T3111C or T257G in the Clock gene with diurnal preference or delayed sleep phase syndrome (DSPS). These controversial data could result from the possible effects of latitude and clock genes interaction on circadian phenotypes.

Increased hypocretin-1 (orexin-a) levels in cerebrospinal fluid of rats after short-term forced activity

The hypocretins (orexins) are recently discovered neuropeptides initially associated with feeding behavior and sleep regulation. However, the normal function of these peptides is unclear and a number of studies have reported a role in energy homeostasis and locomotor activity. Exercise (or physical activity) is the most powerful way of challenging the internal homeostatic process. This study examines the circadian differences in response to forced activity and homeostatic challenges on hypocretin-1 (Hcrt-1) levels in the cerebrospinal fluid (CSF) of rats. Hcrt-1 levels were decreased after long-term immobilization at the end of active phase (zeigeber time-0, ZT-0) and increased after short-term forced swimming in the rest phase (ZT-8). Nevertheless, no effects were observed after short-term immobilization, total sleep deprivation or cold exposure. We concluded that despite the relation between hypocretins, stress and sleep regulation reported in the literature, short-term total sleep deprivation, immobilization and cold exposure did not induce increases in CSF Hcrt-1 levels at ZT-0 and ZT-8. On the other hand, the relationship between hypocretinergic system activation and motor activation is reinforced by decrease in Hcr-1 levels after long-term immobilization at ZT-0 and its increased levels after short-term forced swimming at ZT-8 in CSF of rats.

Increased hypocretin-1 levels in cerebrospinal fluid after REM sleep deprivation

Rat cisternal (CSF) hypocretin-1 in cerebrospinal fluid was measured after 6 or 96 h of REM sleep deprivation and following 24 h of REM sleep rebound. REM deprivation was found to increase CSF hypocretin-1 collected at zeitgeber time (ZT) 8 but not ZT0. Decreased CSF hypocretin levels were also observed at ZT8 after 24 h of REM sleep rebound. These results suggest that REM sleep deprivation activates and REM sleep rebound inhibits the hypocretin system. Increased hypocretin tone during REM deprivation may be important in mediating some of the effects of REM sleep deprivation such as antidepressant effects, hyperphagia and increased sympathetic activity.

Influence of chronotype and social zeitgebers on sleep/wake patterns

Inter-individual differences in the phase of the endogenous circadian rhythms have been established. Individuals with early circadian phase are called morning types; those with late circadian phase are evening types. The Horne and Ostberg Morningness-Eveningness Questionnaire (MEQ) is the most frequently used to assess individual chronotype. The distribution of MEQ scores is likely to be biased by several fact, ors, such as gender, age, genetic background, latitude, and social habits. The objective of the present study was to determine the effect of different social synchronizers on the sleep/wake cycle of persons with different chronotypes. Volunteers were selected from a total of 1232 UFPR undergraduate students who completed the MEQ. Thirty-two subjects completed the study, including 8 morning types, 8 evening types and 16 intermediate types. Sleep schedules were recorded by actigraphy for 1 week on two occasions: during the school term and during vacation. Sleep onset and offset times, sleep duration, and mid-sleep time for each chronotype group were compared by the Mann-Whitney U-test separately for school term and vacation. School term and vacation data were compared by the Wilcoxon matched-pair test. Morning types showed earlier sleep times and longer sleep duration compared with evening types (23:00 +/- 44 and 508.9 +/- 50.27 vs 01:08 +/- 61.95 and 456.44 +/- 59.08, for the weekdays during vacation). During vacation, the subjects showed later sleep times, except for the morning types, who did not exhibit differences for sleep onset times. The results support the idea that social schedules have an impact on the expression of circadian rhythmicity but this impact depends on the individual chronotype.

Nuclear Receptor Rev-Erb-α Circadian Gene Variants and Lithium Carbonate Prophylaxis in Bipolar Affective Disorder

Rev-erb-α is one of the key components of the mammalian circadian mechanism; recently, it was also reported to be involved in the biological action of lithium. We investigated whether polymorphisms in the Rev-erb- α gene are associated with the long-term efficacy of lithium carbonate therapy in bipolar affective disorder. Seven single nucleotide polymorphisms (SNPs) were genotyped in a well-characterized sample of patients from Sardinia, Italy, who were followed prospectively for up to 27 years. Genotypic and allelic analysis did not show evidence for association between the polymorphisms and the different levels of lithium response. Further analyses grouping the different levels of response demonstrated that when the patients were separated into groups of nonresponders versus individuals who have had at least a minor or modest improvement in frequency of episodes or admissions, there was a significant increase in the frequency of the T allele in the nonresponder group (p = 0.0008). Logistic regression analyses showed that patients carrying at least one copy of the T allele for the rs2314339 marker were shown to be approximately 3.5 times more likely to have no improvement or even a worsening of the illness (odds ratio [OR], 3.56; 95% confidence interval [CI], 1.18-10.76). The results of this study may help to identify potential biological markers that can serve to predict the response of bipolar affective disorder patients to treatment, improving treatment efficacy.

Distribution and heritability of diurnal preference (chronotype) in a rural Brazilian family-based cohort, the Baependi study

Diurnal preference (chronotype) is a useful instrument for studying circadian biology in humans. It harbours trait-like dimensions relating to circadian period and sleep homeostasis, but also has ontogenetic components (morningness increases with age). We used the Morningness-Eveningness questionnaire (MEQ) in the Baependi study, a family-based cohort study based in a small town in Minas Gerais, Brazil. The population is highly admixed and has a cohesive and conservative lifestyle. 825 individuals (497 female) aged 18–89 years (average ± SD = 46.4 ± 16.3) and belonging to 112 different families participated in this study. The average MEQ score was 63.5 ± 11.2 with a significant (P < 0.0001) linear increase with age. Morningness was significantly (P < 0.0001) higher in the rural (70.2 ± 9.8) than in the municipal zone (62.6 ± 11.1), and was also significantly (P = 0.025) higher in male (64.6 ± 10.9) than in female (62.8 ± 11.2) participants. Thus, in spite of universal access to electricity, the Baependi population was strongly shifted towards morningness, particularly in the rural zone. Heritability of MEQ score was 0.48 when adjusted for sex and age, or 0.38 when adjusted for sex, age, and residential zone. The reported MEQ score heritability is more akin to those of previous twin studies than previous family studies.

Chronotype ontogeny related to gender

Chronotype is an established concept designed to identify distinct phase relationships between the expression of circadian rhythms and external synchronizers in humans. Although it has been widely accepted that chronotype is subjected to ontogenetic modulation, there is no consensus on the interaction between age and gender. This study aimed to determine the relationship between age- and gender-related changes in the morningness-eveningness character in a large sample of people. A total of 14,650 volunteers were asked to complete the Brazilian version of the Horne and Östberg chronotype questionnaire. The data demonstrated that, on average, women were more morning-oriented than men until the age of 30 and there were no significant differences between men and women from 30 to 45 years of age. In contrast to the situation observed until the age of 30, women older than 45 years were more evening-oriented than men. These results suggest that the ontogenetic development of the circadian timekeeping system is more plastic in men, as represented by the larger amplitude of chronotype changes throughout their aging process. The phase delay of adolescence and phase advance of the elderly seem to be phenomena that are more markedly present in men than in women. Thus, our data, for the first time, provide support that sharply opposes the view that there is a single path toward morningness as a function of age, regardless of gender.

Evolutionary History of the PER3 Variable Number of Tandem Repeats (VNTR): Idiosyncratic Aspect of Primate Molecular Circadian Clock

The PER3 gene is one of the clock genes, which function in the core mammalian molecular circadian system. A variable number of tandem repeats (VNTR) locus in the 18th exon of this gene has been strongly associated to circadian rhythm phenotypes and sleep organization in humans, but it has not been identified in other mammals except primates. To better understand the evolution and the placement of the PER3 VNTR in a phylogenetical context, the present study enlarges the investigation about the presence and the structure of this variable region in a large sample of primate species and other mammals. The analysis of the results has revealed that the PER3 VNTR occurs exclusively in simiiforme primates and that the number of copies of the primitive unit ranges from 2 to 11 across different primate species. Two transposable elements surrounding the 18th exon of PER3 were found in primates with published genome sequences, including the tarsiiforme Tarsius syrichta, which lacks the VNTR. These results suggest that this VNTR may have evolved in a common ancestor of the simiiforme branch and that the evolutionary copy number differentiation of this VNTR may be associated with primate simiiformes sleep and circadian phenotype patterns.

Reciprocal interactions of obstructive sleep apnea and hypertension associated with ACE I/D polymorphism in males

BACKGROUND The angiotensin-converting enzyme (ACE) insertion/deletion (I/D) polymorphism gene contributes to the genesis of hypertension (HTN) and may help explain the relationship between obstructive sleep apnea (OSA) and HTN. However, ACE is a pleiotropic gene that has several influences, including skeletal muscle and control of ventilation. We therefore tested the hypothesis that ACE polymorphism influences OSA severity. METHODS Male OSA patients (apnea-hypopnea index [AHI]>5 events/h) from 2 university sleep centers were evaluated by polysomnography and ACE I/D polymorphism genotyping. RESULTS We studied 266 males with OSA (age=48+/-13 y, body mass index=29+/-5 kg/m(2), AHI=34+/-25 events/h). HTN was present in 114 patients (43%) who were older (p<0.01), heavier (p<0.05) and had more severe OSA (p<0.01). The I allele was associated with HTN in patients with mild to moderate OSA (p<0.01), but not in those with severe OSA. ACE I/D polymorphism was not associated with apnea severity among normotensive patients. In contrast, the only variables independently associated with OSA severity among patients with hypertension in multivariate analysis were BMI (OR=1.12) and II genotype (OR=0.27). CONCLUSIONS Our results indicate reciprocal interactions between OSA and HTN with ACE I/D polymorphism, suggesting that among hypertensive OSA males, the homozygous ACE I allele protects from severe OSA.