Takashi Ebisawa

Genomewide Association Analysis of Human Narcolepsy and a New Resistance Gene

Human narcolepsy is a hypersomnia that is affected by multiple genetic and environmental factors. One genetic factor strongly associated with narcolepsy is the HLA-DRB1*1501-DQB1*0602 haplotype in the human leukocyte antigen region on chromosome 6, whereas the other genetic factors are not clear. To discover additional candidate regions for susceptibility or resistance to human narcolepsy, we performed a genomewide association study, using 23,244 microsatellite markers. Two rounds of screening with the use of pooled DNAs yielded 96 microsatellite markers (including 16 markers on chromosome 6) with significantly different estimated frequencies in case and control pools. Markers not located on chromosome 6 were evaluated by the individual typing of 95 cases and 95 controls; 30 markers still showed significant associations. A strong association was displayed by a marker on chromosome 21 (21q22.3). The surrounding region was subjected to high-density association mapping with 14 additional microsatellite markers and 74 SNPs. One microsatellite marker (D21S0012m) and two SNPs (rs13048981 and rs13046884) showed strong associations (P < .0005; odds ratios 0.19-0.33). These polymorphisms were in a strong linkage disequilibrium, and no other polymorphism in the region showed a stronger association with narcolepsy. The region contains three predicted genes--NLC1-A, NLC1-B, and NLC1-C--tentatively named narcolepsy candidate-region 1 genes, and NLC1-A and NLC1-C were expressed in human hypothalamus. Reporter-gene assays showed that the marker D21S0012m in the promoter region and the SNP rs13046884 in the intron of NLC1-A significantly affected expression levels. Therefore, NLC1-A is considered to be a new resistance gene for human narcolepsy.

Establishment of human cell lines showing circadian rhythms of bioluminescence.

We have established human retinal pigment epithelial cell lines stably expressing the luciferase gene, driven by the human Bmal1 promoter, to obtain human-derived cells that show circadian rhythms of bioluminescence after dexamethasone treatment. The average circadian period of bioluminescence for the obtained clones was 24.07+/-0.48 h. Lithium (10 mM) in the medium significantly lengthened the circadian period of bioluminescence, which is consistent with previous reports, while 2 mM or 5 mM lithium had no effect. This is the first report on the establishment of human-derived cell lines that proliferate infinitely and show circadian rhythms of bioluminescence, and also the first to investigate the effects of low-dose lithium on the circadian rhythms of human-derived cells in vitro. The established cells will be useful for various in vitro studies of human circadian rhythms and for the development of new therapies for human disorders related to circadian rhythm disturbances.

Self-sustained circadian rhythm in cultured human mononuclear cells isolated from peripheral blood

Disturbed circadian rhythmicity is associated with human diseases such as sleep and mood disorders. However, study of human endogenous circadian rhythm is laborious and time-consuming, which hampers the elucidation of diseases. It has been reported that peripheral tissues exhibit circadian rhythmicity as the suprachiasmatic nucleus-the center of the biological clock. We tried to study human circadian rhythm using cultured peripheral blood mononuclear cells (PBMCs) obtained from a single collection of venous blood. Activated human PBMCs showed self-sustained circadian rhythm of clock gene expression, which indicates that they are useful for investigating human endogenous circadian rhythm.

No association of 5-HT2C, 5-HT6, and tryptophan hydroxylase-1 gene polymorphisms with personality traits in the Japanese population.

Serotonin 2C receptor (5-HT(2C)), serotonin 6 receptor (5-HT(6)), and tryptophan hydroxylase-1 (TPH1) genes could be candidates for personality-related genes considering the role of serotonin in various mental functions and behavior. However, a limited number of studies have investigated the association between these genes and personality traits. In the present study, we investigated the three serotonin-related genes, 5-HT(2C), 5-HT(6), and TPH1 genes, in relation to personality traits in the Japanese population. The Cys23Ser polymorphisms in the 5-HT(2C) gene, the 267T/C polymorphism of the 5-HT(6) gene, and the 779A/C polymorphisms in the TPH1 gene were genotyped in 253 healthy Japanese subjects. Personality traits were evaluated by using the Revised NEO Personality Inventory (NEO PI-R) and the State-Trait Anxiety Inventory (STAI). As a result, no significant association was observed between the polymorphisms and the NEO PI-R or the STAI scores. The present results did not provide evidence for the association between the three serotonin-related genes and personality traits. The genes might not have major role in the development of personality traits, although further investigation with larger sample size may be recommended for conclusion.

An association analysis of Per2 with panic disorder in the Japanese population.

Panic disorder (PD) is a severe and chronic psychiatric disorder, with genetic components underlying in its etiology. The PERIOD2 (Per2) gene has been reported to be associated with familial advanced sleep phase syndrome. Considering the high frequency of sleep disturbance in PD, Per2 may be a candidate gene for PD. Therefore, we conducted a two-stage case–control association study in the Japanese population. In the first screening sample of 203 patients and 409 controls, we investigated three single-nucleotide polymorphisms in Per2. We found a potential association in the screening sample (rs2304672, genotype P=0.046, uncorrected), whereas we could not replicate the association in the second sample of 460 patients and 460 controls. Our results suggest that Per2 may not have a major role in the pathogenesis of PD in the Japanese population.

Introduction of tau Mutation into Cultured Rat1-R12 Cells by Gene Targeting, Using Recombinant Adeno-Associated Virus Vector

We aim to develop a cultured cell model, to serve as a system with which the altered circadian phenotypes produced by the clock gene variations could be studied in vitro. Tau mutation, which shortens the circadian period of hamsters and mice, was introduced into the CK1ε locus of cultured Rat1-R12 cells by gene targeting mediated by a recombinant adeno-associated virus (rAAV) vector. After transduction of Rat1-R12 cells with rAAV, about 0.14% of the drug-resistant cells underwent gene targeting at CK1ε locus. Of the three clones isolated, only one carried the targeted allele of tau mutation and two carried the targeted wild-type allele. The clone with the targeted tau mutant allele exhibited a significantly shorter circadian period compared to the clone with targeted wild-type allele. rAAV-mediated gene targeting in cultured somatic cells is a convenient and powerful tool for analyzing the phenotypic outcome of clock gene variations, and for elucidating the pathogenesis of the disorders associated with abnormal circadian rhythmicity.

No association between CLOCK gene 3111C/T polymorphism and personality traits in healthy Japanese subjects

THE CLOCK GENE 3111 C/T polymorphism has been reported to be associated with human diurnal preference and psychiatric disorders, including schizophrenia and bipolar disorder. Furthermore, specific personality traits may be correlated with psychiatric symptoms. Therefore, we hypothesized that the CLOCK gene could be a candidate for the personality-related gene and investigated the association between CLOCK and personality traits in Japanese healthy subjects. The research protocol was approved by the Ethics Committee of the Graduate School of Medicine, University of Tokyo. All participants provided written informed consent. All participants were unrelated Japanese subjects. The subjects consisted of 383 healthy volunteers (139 men and 244 women; mean age 35.4 11.2 years [mean SD]) recruited from the staff of several mental and general hospitals around Tokyo. We genotyped the CLOCK gene 3111C/T polymorphism (rs1801260) using the TaqMan assay (Applied Biosystems, Foster, CA, USA). Personality traits were evaluated by using the Revised NEO Personality Inventory (NEO PI-R). The associations between scores for the NEO PI-R factors and genotypes were statistically analyzed by ANCOVA with sex and age as covariates. The genotype distributions did not deviate significantly from Hardy–Weinberg equilibrium. Because the frequency of the homozygotes for the C allele of CLOCK was very low, we also compared scores for the NEO PI-R factors between T/T genotype and C allele carrier (C/T and C/C genotypes) groups. No significant difference in the scores for the NEO PI-R factors was observed among the three genotype groups or between T/T genotype and C allele carrier groups (Table 1). Our study did not provide evidence for the association between the CLOCK gene 3111 C/T polymorphism and personality traits in the Japanese population. REFERENCES

Gene analysis for circadian-based sleep disorders

Approximately 50% of all discordance in monozygotic (MZ) twins for schizophrenia has been attributed to penetrancc, an unexplained genetic phenomenon. A mechanism for the discordance, if it is revealed, may provide a clue to the primary prevention of schizophrenia. We applied restriction landmark genome scanning (RLGS) ver.1.X (Okazaki et al., 1003) using Not1 as a restriction enzyme to be radio-labeled to a MZ twin discordant for schizophrenia, assuming that there is a genomic mechanism for the development of discordance. We have found at least two discordant RLGS spots between affected and non-affected twins (Tsujita et al., 19%). Epigenetic differences in the methylation status of CpG islands near the promoters of genes are considered to be the most probable cause of these discordant RLGS spots according to the nature of NotI. Direct cloning of DNA fragments from the discordant spots may lead to the elucidation of schizophrcniarelated genes as well as a genomic mechanism of discordance or penetrance in MZ. This method of analysis is available for not only schizophrenia but also other multifactorial disorders such as bipolar and panic dicorders.