Yuji Ozeki

Association of structural polymorphisms in the human period3 gene with delayed sleep phase syndrome

Recent progress in biological clock research has facilitated genetic analysis of circadian rhythm sleep disorders, such as delayed sleep phase syndrome (DSPS) and non‐24‐h sleep–wake syndrome (N‐24). We analyzed the human period3 (hPer3) gene, one of the human homologs of the Drosophila clock‐gene period (Per), as a possible candidate for rhythm disorder susceptibility. All of the coding exons in the hPer3 gene were screened for polymorphisms by a PCR‐based strategy using genomic DNA samples from sleep disorder patients and control subjects. We identified six sequence variations with amino acid changes, of which five were common and predicted four haplotypes of the hPer3 gene. One of the haplotypes was significantly associated with DSPS (Bonferronis corrected P = 0.037; odds ratio = 7.79; 95% CI 1.59–38.3) in our study population. Our results suggest that structural polymorphisms in the hPer3 gene may be implicated in the pathogenesis of DSPS.

Disrupted-in-Schizophrenia-1 (DISC-1): Mutant truncation prevents binding to NudE-like (NUDEL) and inhibits neurite outgrowth

Disrupted-in-Schizophrenia-1 (DISC-1) is a gene whose mutant truncation is associated with major psychiatric illness with a predominance of schizophrenic symptomatology. We have cloned and characterized rodent DISC-1. DISC-1 expression displays pronounced developmental regulation with the highest levels in late embryonic life when the cerebral cortex develops. In yeast two-hybrid analyses, DISC-1 interacts with a variety of cytoskeletal proteins. One of these, NudE-like (NUDEL), is associated with cortical development and is linked to LIS-1, the disease gene for a form of lissencephaly, a disorder of cortical development. The disease mutant form of DISC-1 fails to bind NUDEL. Expression of mutant, but not wild-type, DISC-1 in PC12 cells reduces neurite extension. As schizophrenia is thought to reflect defects in cortical development that are determined by cytoskeletal protein activities, the cellular disturbances we observe with mutant DISC-1 may be relevant to psychopathologic mechanisms.

Minimum light intensity required to suppress nocturnal melatonin concentration in human saliva

We set out to determine the minimum intensity of light able to suppress nocturnal melatonin levels as measured in normal human saliva. Five healthy male volunteers were exposed to light at different intensities (<10, 500, 1000, 2500, and 5000 lux) in a repeated measure design. Suppression of melatonin was dependent on both light intensity and duration of light exposure. Minimum intensities of light suppressing nocturnal melatonin levels were calculated as 393, 366, 339, and 285 lux for exposure durations of 30, 60, 90, and 120 min, respectively. Minimum effective intensity and duration of light exposure showed a linear inverse relationship. These results suggest that less intensity of light than previously reported suffices to suppress melatonin in humans, and that caution is required in interpreting studies using long exposure to dim light as a background condition.

HYPERSENSITIVITY OF MELATONIN SUPPRESSION IN RESPONSE TO LIGHT IN PATIENTS WITH DELAYED SLEEP PHASE SYNDROME

Patients with delayed sleep phase syndrome (DSPS) experiencea chronic mismatch between the usual daily schedule required by the individualsenvironment and their circadian sleep-wake pattern, resulting in major academic,work, and social problems. Although functional abnormalities of the circadianpacemaker system have been reported in patients with DSPS, the etiology ofDSPS has not been fully elucidated. One hypothesis proposed to explain whypatients with DSPS fail to synchronize their 24h sleep-wake cycle to theirenvironment is that they might have reduced sensitivity to environmental timecues, most notably light-dark cycles. Therefore, we compared the sensitivityof melatonin suppression in response to light in patients with DSPS and normalcontrol subjects. Fifteen patients with DSPS and age- and sex-matched healthycontrols were studied. As the melatonin secretion rhythm in patients withDSPS was expected to be delayed compared to the controls, the time of peakmelatonin secretion was determined in each subject in the first session. Inthe second session, each subject was exposed to light with an intensity of1000 lux for 2h beginning 2h prior to his or her peak melatonin secretion.Melatonin was measured by radioimmunoassay in saliva sampled every 30 minutesduring the period of light exposure. Suppression of the melatonin concentrationin saliva was dependent on duration of light exposure. In addition, the suppressiveeffect of light on the melatonin concentration was significantly greater inpatients with DSPS than in control subjects. The results suggest hypersensitivityto nighttime light exposure in patients with this syndrome. Our findings thereforesuggest that evening light restriction is important for preventing patientswith DSPS from developing a sleep phase delay. (ChronobiologyInternational, 18(2), 263–271, 2001)

Genetic polymorphisms of human melatonin 1b receptor gene in circadian rhythm sleep disorders and controls

Recent studies suggest that melatonin 1b (Mel1b) receptor, as well as melatonin 1a (Mel1a) receptor, is involved in the modulation of circadian rhythms in mammals. Mutational analysis was performed in the entire coding region of the human Mel1b receptor gene using genomic DNA from sleep disorder subjects. We have identified two missense mutations, G24E and L66F. However, neither is likely to be associated with sleep disorders in our study population. One of the subjects with non-24-h sleep-wake syndrome carries missense mutations in both the Mel1a and Mel1b receptor genes.

Mutation screening of the human period 2 gene in bipolar disorder

We tested whether the human period 2 gene (hper2), one of the essential components of the circadian oscillator, might have influence on bipolar disorder. We screened 88 bipolar disorder patients and 127 controls, all of Japanese origin. Screening in the casein kinase I epsilon (CKIepsilon) binding region of hper2, which was previously reported in familial advanced sleep-phase syndrome patients, with polymerase chain reaction amplification revealed four polymorphisms. One of the four polymorphisms had an amino acid substitution of a serine at 662 with a glycine (S662G). The frequencies of the S662G allele and genotypes on patients with bipolar disorder were very low and had no difference from those in controls. Polymorphism on the CKIepsilon binding region of hper2 gene which was previously reported, is unlikely to play an important role in the development of bipolar disorder.

A newly developed assay for melatonin using cells expressing human mel-1a receptor.

We have developed a radioreceptor binding assay (RRA) method for melatonin using membranes from Chinese hamster ovary cells that can stably express human mel‐1a receptors. We measured melatonin levels in plasma samples collected every 4 h for 24 h using the RRA and radioimmunoassay (RIA) methods, simultaneously. There was a statistically significant correlation between the melatonin levels measured by the two methods, this newly developed method providing a sensitive bioassay. As it is possible to circumvent the cross‐reactivity usually occurring in the RIA method, this method may be an important tool for detecting bioactive substances relative to the mel‐1a receptor.