Genome-wide association study of idiopathic hypersomnia in a Japanese population.

Idiopathic hypersomnia (IH) is a rare sleep disorder characterized by excessive daytime sleepiness, great difficulty upon awakening, and prolonged sleep time. In contrast to narcolepsy type 1, which is a well-recognized hypersomnia, the etiology of IH remains poorly understood. No susceptibility loci for IH have been identified, although familial aggregations have been observed among patients with IH. Narcolepsy type 1 is strongly associated with human leukocyte antigen (HLA)-DQB1*06:02; however, no significant associations between IH and HLA alleles have been reported. To identify genetic variants that affect susceptibility to IH, we performed a genome-wide association study (GWAS) and two replication studies involving a total of 414 Japanese patients with IH and 6587 healthy Japanese individuals. A meta-analysis of the three studies found no single-nucleotide polymorphisms (SNPs) that reached the genome-wide significance level. However, we identified several candidate SNPs for IH. For instance, a common genetic variant (rs2250870) within an intron of PDE9A was suggestively associated with IH. rs2250870 was significantly associated with expression levels of PDE9A in not only whole blood but also brain tissues. The leading SNP in the PDE9A region was the same in associations with both IH and PDE9A expression. PDE9A is a potential target in the treatment of several brain diseases, such as depression, schizophrenia, and Alzheimer's disease. It will be necessary to examine whether PDE9A inhibitors that have demonstrated effects on neurophysiologic and cognitive function can contribute to the development of new treatments for IH, as higher expression levels of PDE9A were observed with regard to the risk allele of rs2250870. The present study constitutes the first GWAS of genetic variants associated with IH. A larger replication study will be required to confirm these associations. Supplementary Information: The online version contains supplementary material available at 10.1007/s41105-021-00349-2.

Metabolome analysis using cerebrospinal fluid from narcolepsy type 1 patients.

Narcolepsy type 1 (NT1) is a hypersomnia characterized by excessive daytime sleepiness and cataplexy. Inappropriate regulation of fatty acid metabolism has been suggested to be involved in the pathophysiology of NT1, but the detailed mechanisms remain uncertain. Here we performed a metabolomic analysis of cerebrospinal fluid samples from 14 NT1 and 17 control subjects using a novel capillary electrophoresis coupled with Fourier transform mass spectrometry. A total of 268 metabolites were identified and the amount of histidine was the most significantly increased in NT1 patients (p = 4.0 × 10-4). Validation analysis using high-performance liquid chromatography (HPLC) including independent replication samples also identified the association of histidine (p = 2.02 × 10-3). Further, levels of histamine, which is synthesized from histidine, were also examined using HPLC and were found to be significantly decreased in NT1 patients (p = 6.12 × 10-4). Pathway analysis with nominally significant metabolites identified several pathways related to the metabolism of glycogenic amino acids, suggesting that glycogenesis is enhanced in NT1 as a compensatory mechanism for fatty acid metabolism. We performed further exploratory analysis, searching for metabolites associated with sleep variables from polysomnography and the multiple sleep latency test. As a result, 5'-deoxy-5'-methylthioadenosine showed a significant association with apnea-hypopnea index (p = 2.66 ×10-6). Moreover, gamma aminobutyric acid displayed a negative correlation with rapid eye movement sleep latency (REML), and thus might represent an intriguing target for future studies to elucidate how the controlling circuit of REM sleep is associated with abnormally short REML in NT1.

Sensitivity to gene dosage and gene expression affects genes with copy number variants observed among neuropsychiatric diseases.

BACKGROUND: Copy number variants (CNVs) have been reported to be associated with diseases, traits, and evolution. However, it is hard to determine which gene should have priority as a target for further functional experiments if a CNV is rare or a singleton. In this study, we attempted to overcome this issue by using two approaches: by assessing the influences of gene dosage sensitivity and gene expression sensitivity. Dosage sensitive genes derived from two-round whole-genome duplication in previous studies. In addition, we proposed a cross-sectional omics approach that utilizes open data from GTEx to assess the effect of whole-genome CNVs on gene expression. METHODS: Affymetrix Genome-Wide SNP Array 6.0 was used to detect CNVs by PennCNV and CNV Workshop. After quality controls for population stratification, family relationship and CNV detection, 287 patients with narcolepsy, 133 patients with essential hypersomnia, 380 patients with panic disorders, 164 patients with autism, 784 patients with Alzheimer disease and 1280 healthy individuals remained for the enrichment analysis. RESULTS: Overall, significant enrichment of dosage sensitive genes was found across patients with narcolepsy, panic disorders and autism. Particularly, significant enrichment of dosage-sensitive genes in duplications was observed across all diseases except for Alzheimer disease. For deletions, less or no enrichment of dosage-sensitive genes with deletions was seen in the patients when compared to the healthy individuals. Interestingly, significant enrichments of genes with expression sensitivity in brain were observed in patients with panic disorder and autism. While duplications presented a higher burden, deletions did not cause significant differences when compared to the healthy individuals. When we assess the effect of sensitivity to genome dosage and gene expression at the same time, the highest ratio of enrichment was observed in the group including dosage-sensitive genes and genes with expression sensitivity only in brain. In addition, shared CNV regions among the five neuropsychiatric diseases were also investigated. CONCLUSIONS: This study contributed the evidence that dosage-sensitive genes are associated with CNVs among neuropsychiatric diseases. In addition, we utilized open data from GTEx to assess the effect of whole-genome CNVs on gene expression. We also investigated shared CNV region among neuropsychiatric diseases.

Epigenome-wide association study of narcolepsy-affected lateral hypothalamic brains, and overlapping DNA methylation profiles between narcolepsy and multiple sclerosis.

Narcolepsy with cataplexy is a sleep disorder caused by a deficiency in hypocretin neurons in the lateral hypothalamus (LH). Here we performed an epigenome-wide association study (EWAS) of DNA methylation for narcolepsy and replication analyses using DNA samples extracted from two brain regions: LH (Cases: N = 4; Controls: N = 4) and temporal cortex (Cases: N = 7; Controls: N = 7). Seventy-seven differentially methylated regions (DMRs) were identified in the LH analysis, with the top association of a DMR in the myelin basic protein (MBP) region. Only five DMRs were detected in the temporal cortex analysis. Genes annotated to LH DMRs were significantly associated with pathways related to fatty acid response or metabolism. Two additional analyses applying the EWAS data were performed: (1) investigation of methylation profiles shared between narcolepsy and other disorders and (2) an integrative analysis of DNA methylation data and a genome-wide association study for narcolepsy. The results of the two approaches, which included significant overlap of methylated positions associated with narcolepsy and multiple sclerosis, indicated that the two diseases may partly share their pathogenesis. In conclusion, DNA methylation in LH where loss of orexin-producing neurons occurs may play a role in the pathophysiology of the disease.

A variant at 9q34.11 is associated with HLA-DQB1*06:02 negative essential hypersomnia.

Essential hypersomnia (EHS) is a lifelong disorder characterized by excessive daytime sleepiness without cataplexy. EHS is associated with human leukocyte antigen (HLA)-DQB1*06:02, similar to narcolepsy with cataplexy (narcolepsy). Previous studies suggest that DQB1*06:02-positive and -negative EHS are different in terms of their clinical features and follow different pathological pathways. DQB1*06:02-positive EHS and narcolepsy share the same susceptibility genes. In the present study, we report a genome-wide association study with replication for DQB1*06:02-negative EHS (408 patients and 2247 healthy controls, all Japanese). One single-nucleotide polymorphism, rs10988217, which is located 15-kb upstream of carnitine O-acetyltransferase (CRAT), was significantly associated with DQB1*06:02-negative EHS (P = 7.5 × 10-9, odds ratio = 2.63). The risk allele of the disease-associated SNP was correlated with higher expression levels of CRAT in various tissues and cell types, including brain tissue. In addition, the risk allele was associated with levels of succinylcarnitine (P = 1.4 × 10-18) in human blood. The leading SNP in this region was the same in associations with both DQB1*06:02-negative EHS and succinylcarnitine levels. The results suggest that DQB1*06:02-negative EHS may be associated with an underlying dysfunction in energy metabolic pathways.

Epigenome-wide association study of DNA methylation in narcolepsy: an integrated genetic and epigenetic approach.

Narcolepsy with cataplexy, which is a hypersomnia characterized by excessive daytime sleepiness and cataplexy, is a multifactorial disease caused by both genetic and environmental factors. Several genetic factors including HLA-DQB1*06:02 have been identified; however, the disease etiology is still unclear. Epigenetic modifications, such as DNA methylation, have been suggested to play an important role in the pathogenesis of complex diseases. Here, we examined DNA methylation profiles of blood samples from narcolepsy and healthy control individuals and performed an epigenome-wide association study (EWAS) to investigate methylation loci associated with narcolepsy. Moreover, data from the EWAS and a previously performed narcolepsy genome-wide association study were integrated to search for methylation loci with causal links to the disease. We found that (1) genes annotated to the top-ranked differentially methylated positions (DMPs) in narcolepsy were associated with pathways of hormone secretion and monocarboxylic acid metabolism. (2) Top-ranked narcolepsy-associated DMPs were significantly more abundant in non-CpG island regions and more than 95 per cent of such sites were hypomethylated in narcolepsy patients. (3) The integrative analysis identified the CCR3 region where both a single methylation site and multiple single-nucleotide polymorphisms were found to be associated with the disease as a candidate region responsible for narcolepsy. The findings of this study suggest the importance of future replication studies, using methylation technologies with wider genome coverage and/or larger number of samples, to confirm and expand on these results.

Narcolepsy susceptibility gene CCR3 modulates sleep-wake patterns in mice.

Narcolepsy is caused by the loss of hypocretin (Hcrt) neurons and is associated with multiple genetic and environmental factors. Although abnormalities in immunity are suggested to be involved in the etiology of narcolepsy, no decisive mechanism has been established. We previously reported chemokine (C-C motif) receptor 3 (CCR3) as a novel susceptibility gene for narcolepsy. To understand the role of CCR3 in the development of narcolepsy, we investigated sleep-wake patterns of Ccr3 knockout (KO) mice. Ccr3 KO mice exhibited fragmented sleep patterns in the light phase, whereas the overall sleep structure in the dark phase did not differ between Ccr3 KO mice and wild-type (WT) littermates. Intraperitoneal injection of lipopolysaccharide (LPS) promoted wakefulness and suppressed both REM and NREM sleep in the light phase in both Ccr3 KO and WT mice. Conversely, LPS suppressed wakefulness and promoted NREM sleep in the dark phase in both genotypes. After LPS administration, the proportion of time spent in wakefulness was higher, and the proportion of time spent in NREM sleep was lower in Ccr3 KO compared to WT mice only in the light phase. LPS-induced changes in sleep patterns were larger in Ccr3 KO compared to WT mice. Furthermore, we quantified the number of Hcrt neurons and found that Ccr3 KO mice had fewer Hcrt neurons in the lateral hypothalamus compared to WT mice. We found abnormalities in sleep patterns in the resting phase and in the number of Hcrt neurons in Ccr3 KO mice. These observations suggest a role for CCR3 in sleep-wake regulation in narcolepsy patients.

Hypocretin/orexin loss changes the hypothalamic immune response.

Hypocretin, also known as orexin, maintains the vigilance state and regulates various physiological processes, such as arousal, sleep, food intake, energy expenditure, and reward. Previously, we found that when wild-type mice and hypocretin/ataxin-3 littermates (which are depleted of hypothalamic hypocretin-expressing neurons postnatally) were administered lipopolysaccharide (LPS), the two genotypes exhibited significant differences in their sleep/wake cycle, including differences in the degree of increase in sleep periods and in recovery from sickness behaviour. In the present study, we examined changes in the hypothalamic vigilance system and in the hypothalamic expression of inflammatory factors in response to LPS in hypocretin/ataxin-3 mice. Peripheral immune challenge with LPS affected the hypothalamic immune response and vigilance states. This response was altered by the loss of hypocretin. Hypocretin expression was inhibited after LPS injection in both hypocretin/ataxin-3 mice and their wild-type littermates, but expression was completely abolished only in hypocretin/ataxin-3 mice. Increases in the number of histidine decarboxylase (HDC)-positive cells and in Hdc mRNA expression were found in hypocretin/ataxin-3 mice, and this increase was suppressed by LPS. Hypocretin loss did not impact the change in expression of hypothalamic inflammatory factors in response to LPS, except for interferon gamma and colony stimulating factor 3. The number of c-Fos-positive/HDC-positive cells in hypocretin/ataxin-3 mice administered LPS injections was elevated, even during the rest period, in all areas, suggesting that there is an increase in the activity of histaminergic neurons in hypocretin/ataxin-3 mice following LPS injection. Taken together, our results suggest a novel role for hypocretin in the hypothalamic response to peripheral immune challenge. Our findings contribute to the understanding of the pathophysiology of narcolepsy.

Evaluation of polygenic risks for narcolepsy and essential hypersomnia.

In humans, narcolepsy is a sleep disorder that is characterized by sleepiness, cataplexy and rapid eye movement (REM) sleep abnormalities. Essential hypersomnia (EHS) is another type of sleep disorder that is characterized by excessive daytime sleepiness without cataplexy. A human leukocyte antigen (HLA) class II allele, HLA-DQB1*06:02, is a major genetic factor for narcolepsy. Almost all narcoleptic patients are carriers of this HLA allele, while 30-50% of EHS patients and 12% of all healthy individuals in Japan carry this allele. The pathogenesis of narcolepsy and EHS is thought to be partially shared. To evaluate the contribution of common single-nucleotide polymorphisms (SNPs) to narcolepsy onset and to assess the common genetic background of narcolepsy and EHS, we conducted a polygenic analysis that included 393 narcoleptic patients, 38 EHS patients with HLA-DQB1*06:02, 119 EHS patients without HLA-DQB1*06:02 and 1582 healthy individuals. We also included 376 individuals with panic disorder and 213 individuals with autism to confirm whether the results were biased. Polygenic risks in narcolepsy were estimated to explain 58.1% (PHLA-DQB1*06:02=2.30 × 10-48, Pwhole genome without HLA-DQB1*06:02=6.73 × 10-2) including HLA-DQB1*06:02 effects and 1.3% (Pwhole genome without HLA-DQB1*06:02=2.43 × 10-2) excluding HLA-DQB1*06:02 effects. The results also indicated that small-effect SNPs contributed to the development of narcolepsy. Reported susceptibility SNPs for narcolepsy in the Japanese population, CPT1B (carnitine palmitoyltransferase 1B), TRA@ (T-cell receptor alpha) and P2RY11 (purinergic receptor P2Y, G-protein coupled, 11), were found to explain 0.8% of narcolepsy onset (Pwhole genome without HLA-DQB1*06:02=9.74 × 10-2). EHS patients with HLA-DQB1*06:02 were estimated to have higher shared genetic background to narcoleptic patients than EHS patients without HLA-DQB1*06:02 even when the effects of HLA-DQB1*06:02 were excluded (EHS with HLA-DQB1*06:02: 40.4%, PHLA-DQB1*06:02=7.02 × 10-14, Pwhole genome without HLA-DQB1*06:02=1.34 × 10-1, EHS without HLA-DQB1*06:02: 0.4%, Pwhole genome without HLA-DQB1*06:02=3.06 × 10-1). Meanwhile, the polygenic risks for narcolepsy could not explain the onset of panic disorder and autism, suggesting that our results were reasonable.

Hypocretin/orexin prevents recovery from sickness.

Sickness behavior is defined as states of lethargy, depression, anxiety, loss of appetite, hypersomnia, hyperalgesia, reduction of grooming and failure to concentrate that can be induced by inflammatory diseases, such as infections and cancer. Recent findings revealed that the lipopolysaccharide (LPS) injection causes lethargy as a consequence of the inhibition of hypocretin signaling. The hypocretin system maintains the vigilance state in various physiological processes. In order to investigate the sleep arousal system against sickness behavior, LPS-induced sickness behavior was examined in hypocretin-ataxin-3 transgenic mice, whose hypocretin neurons were postnatally ablated. Sleep-wake activity was determined following the administration of LPS at Zeitgeber time (ZT) 8.0 in ataxin-3 transgenic mice, and the age-, gender-matched wild-type littermates. LPS injection induced increases in non-rapid eye movement (REM) sleep in the matched wild-type littermates. In addition, a further increase in periods of sleep according to the loss of hypocretin neurons was identified in the ataxin-3 transgenic mice. A marked reduction of awakening during ZT12-ZT18 was observed as expected following LPS injection in the mouse lines. The increase in the period of non-REM sleep was not observed on the next day following LPS administration in either of the mouse lines. Complete recovery of physical activity was not observed in the matched wild-type littermates. Ataxin-3 transgenic mice recovered their physical activity to the same level as that on the first day before LPS administration. These results suggest the possibility that a faster recovery is the result of deeper resting according to the absence of hypocretin neurons, as ataxin-3 transgenic mice demonstrated more non-REM sleep.

A polymorphism in CCR1/CCR3 is associated with narcolepsy.

Etiology of narcolepsy-cataplexy involves multiple genetic and environmental factors. While the human leukocyte antigen (HLA)-DRB1*15:01-DQB1*06:02 haplotype is strongly associated with narcolepsy, it is not sufficient for disease development. To identify additional, non-HLA susceptibility genes, we conducted a genome-wide association study (GWAS) using Japanese samples. An initial sample set comprising 409 cases and 1562 controls was used for the GWAS of 525,196 single nucleotide polymorphisms (SNPs) located outside the HLA region. An independent sample set comprising 240 cases and 869 controls was then genotyped at 37 SNPs identified in the GWAS. We found that narcolepsy was associated with a SNP in the promoter region of chemokine (C-C motif) receptor 1 (CCR1) (rs3181077, P=1.6×10(-5), odds ratio [OR]=1.86). This rs3181077 association was replicated with the independent sample set (P=0.032, OR=1.36). We measured mRNA levels of candidate genes in peripheral blood samples of 38 cases and 37 controls. CCR1 and CCR3 mRNA levels were significantly lower in patients than in healthy controls, and CCR1 mRNA levels were associated with rs3181077 genotypes. In vitro chemotaxis assays were also performed to measure monocyte migration. We observed that monocytes from carriers of the rs3181077 risk allele had lower migration indices with a CCR1 ligand. CCR1 and CCR3 are newly discovered susceptibility genes for narcolepsy. These results highlight the potential role of CCR genes in narcolepsy and support the hypothesis that patients with narcolepsy have impaired immune function.

An association analysis of HLA-DQB1 with narcolepsy without cataplexy and idiopathic hypersomnia with/without long sleep time in a Japanese population.

Narcolepsy without cataplexy (NA w/o CA) (narcolepsy type 2) is a lifelong disorder characterized by excessive daytime sleepiness and rapid eye movement (REM) sleep abnormalities, but no cataplexy. In the present study, we examined the human leukocyte antigen HLA-DQB1 in 160 Japanese patients with NA w/o CA and 1,418 control subjects. Frequencies of DQB1*06:02 were significantly higher in patients with NA w/o CA compared with controls (allele frequency: 16.6 vs. 7.8%, P=1.1×10(-7), odds ratio (OR)=2.36; carrier frequency: 31.3 vs. 14.7%, P=7.6×10(-8), OR=2.64). Distributions of HLA-DQB1 alleles other than DQB1*06:02 were compared between NA w/o CA and narcolepsy with cataplexy (NA-CA) to assess whether the genetic backgrounds of the two diseases have similarities. The distribution of the HLA-DQB1 alleles in DQB1*06:02-negative NA w/o CA was significantly different from that in NA-CA (P=5.8×10(-7)). On the other hand, the patterns of the HLA-DQB1 alleles were similar between DQB1*06:02-positive NA w/o CA and NA-CA. HLA-DQB1 analysis was also performed in 186 Japanese patients with idiopathic hypersomnia (IHS) with/without long sleep time, but no significant associations were observed.

New susceptibility variants to narcolepsy identified in HLA class II region.

Narcolepsy, a sleep disorder characterized by excessive daytime sleepiness, cataplexy and rapid eye movement sleep abnormalities, is tightly associated with human leukocyte antigen HLA-DQB1*06:02. DQB1*06:02 is common in the general population (10-30%); therefore, additional genetic factors are needed for the development of narcolepsy. In the present study, HLA-DQB1 in 664 Japanese narcoleptic subjects and 3131 Japanese control subjects was examined to determine whether HLA-DQB1 alleles located in trans of DQB1*06:02 are associated with narcolepsy. The strongest association was with DQB1*06:01 (P = 1.4 × 10(-10), odds ratio, OR = 0.39), as reported in previous studies. Additional predisposing effects of DQB1*03:02 were also found (P = 2.5 × 10(-9), OR = 1.97). A comparison between DQB1*06:02 heterozygous cases and controls revealed dominant protective effects of DQB1*06:01 and DQB1*05:01. In addition, a single-nucleotide polymorphism-based conditional analysis controlling for the effect of HLA-DQB1 was performed to determine whether there were other independent HLA associations outside of HLA-DQB1. This analysis revealed associations at HLA-DPB1 in the HLA class II region (rs3117242, P = 4.1 × 10(-5), OR = 2.45; DPB1*05:01, P = 8.1 × 10(-3), OR = 1.39). These results indicate that complex HLA class II associations contribute to the genetic predisposition to narcolepsy.

Increased plasma IL-6, IL-8, TNF-alpha, and G-CSF in Japanese narcolepsy.

Narcolepsy is a chronic hypersomnia involving excessive daytime sleepiness and cataplexy. Some susceptibility genes and environmental factors suggest that post-infectious immunological alterations underlie its pathophysiology. To investigate the immunological alterations in narcolepsy patients, we examined cytokines. Nine healthy controls and twenty-one narcolepsy patients with cataplexy were studied. All subjects were positive for the HLA-DRB1(∗)1501-DQB1(∗)0602 allele. Age-, sex-, and body mass index -matched healthy controls were selected. Plasma samples were separated using EDTA-2K-coated blood collection tubes. Bioplex Pro Human Cytokine 17-Plex Assays were used to measure plasma cytokines. Elevations of interleukin (IL)-6, IL-8, granulocyte- colony stimulating factor (G-CSF), and tumor necrosis factor-alpha were found in the narcolepsy group compared with healthy controls (p<0.05). G-CSF values were significantly correlated with the disease duration in narcolepsy patients (r=0.426, p<0.05). IL-8 and G-CSF play major roles in neutrophil activation in respiratory diseases. Since environmental factors including infection are reportedly associated with narcolepsy onset, elevated IL-8 and G-CSF may be involved in the pathophysiology of narcolepsy.

Genome-wide analysis of CNV (copy number variation) and their associations with narcolepsy in a Japanese population.

In humans, narcolepsy with cataplexy (narcolepsy) is a sleep disorder that is characterized by sleepiness, cataplexy and rapid eye movement (REM) sleep abnormalities. Narcolepsy is caused by a reduction in the number of neurons that produce hypocretin (orexin) neuropeptide. Both genetic and environmental factors contribute to the development of narcolepsy.Rare and large copy number variations (CNVs) reportedly play a role in the etiology of a number of neuropsychiatric disorders. Narcolepsy is considered a neurological disorder; therefore, we sought to investigate any possible association between rare and large CNVs and human narcolepsy. We used DNA microarray data and a CNV detection software application, PennCNV-Affy, to detect CNVs in 426 Japanese narcoleptic patients and 562 healthy individuals. Overall, we found a significant enrichment of rare and large CNVs (frequency ≤1%, size ≥100 kb) in the patients (case-control ratio of CNV count=1.54, P=5.00 × 10(-4)). Next, we extended a region-based association analysis by including CNVs with its size ≥30 kb. Rare and large CNVs in PARK2 region showed a significant association with narcolepsy. Four patients were assessed to carry duplications of the gene region, whereas no controls carried the duplication, which was further confirmed by quantitative PCR assay. This duplication was also found in 2 essential hypersomnia (EHS) patients out of 171 patients. Furthermore, a pathway analysis revealed enrichments of gene disruptions by rare and large CNVs in immune response, acetyltransferase activity, cell cycle regulation and regulation of cell development. This study constitutes the first report on the risk association between multiple rare and large CNVs and the pathogenesis of narcolepsy. In the future, replication studies are needed to confirm the associations.

Genome-wide association study of HLA-DQB1*06:02 negative essential hypersomnia.

Essential hypersomnia (EHS), a sleep disorder characterized by excessive daytime sleepiness, can be divided into two broad classes based on the presence or absence of the HLA-DQB1*06:02 allele. HLA-DQB1*06:02-positive EHS and narcolepsy with cataplexy are associated with the same susceptibility genes. In contrast, there are fewer studies of HLA-DQB1*06:02 negative EHS which, we hypothesized, involves a different pathophysiological pathway than does narcolepsy with cataplexy. In order to identify susceptibility genes associated with HLA-DQB1*06:02 negative EHS, we conducted a genome-wide association study (GWAS) of 125 unrelated Japanese EHS patients lacking the HLA-DQB1*06:02 allele and 562 Japanese healthy controls. A comparative study was also performed on 268 HLA-DQB1*06:02 negative Caucasian hypersomnia patients and 1761 HLA-DQB1*06:02 negative Caucasian healthy controls. We identified three SNPs that each represented a unique locus- rs16826005 (P = 1.02E-07; NCKAP5), rs11854769 (P = 6.69E-07; SPRED1), and rs10988217 (P = 3.43E-06; CRAT) that were associated with an increased risk of EHS in this Japanese population. Interestingly, rs10988217 showed a similar tendency in its association with both HLA-DQB1*06:02 negative EHS and narcolepsy with cataplexy in both Japanese and Caucasian populations. This is the first GWAS of HLA-DQB1*06:02 negative EHS, and the identification of these three new susceptibility loci should provide additional insights to the pathophysiological pathway of this condition.

Transcriptional regulation of the hypocretin/orexin gene by NR6A1.

The hypocretin (also known as orexin) neuropeptide system coordinates the regulation of various physiological processes. A reduction in Nr6a1 expression was observed in hypocretin neuron-ablated transgenic mice. To show that prepro-hypocretin transcription is functionally modulated by NR6A1, we performed chromatin immunoprecipitation (ChIP) analysis, double-immunostaining, a luciferase reporter assay, and an in utero electroporation study. ChIP analysis showed that endogenous NR6A1 binds to a putative NR6A1-binding site. Double-immunostaining indicated almost all hypocretin neurons were positive for NR6A1 immunoreactivity. NR6A1 overexpression in SH-SY5Y cells modulated hypocretin promoter activity, an effect that was countered by lacking a putative NR6A1-binding site. Electroporation with Nr6a1 in the foetal hypothalamus promoted hypocretin transcription as compared to GFP-electroporation. These experiments confirmed that NR6A1 works as a regulator for hypocretin transcription.

Anti-Tribbles homolog 2 autoantibodies in Japanese patients with narcolepsy.

STUDY OBJECTIVES: Narcolepsy is a sleep disorder characterized by excessive daytime sleepiness and cataplexy. The association with human leukocyte antigen (HLA)-DQB1*0602 and T-cell receptor alpha locus suggests that autoimmunity plays a role in narcolepsy. A recent study reported an increased prevalence of autoantibodies against Tribbles homolog 2 (TRIB2) in patients with narcolepsy. To replicate this finding, we examined anti-TRIB2 autoantibodies in Japanese patients with narcolepsy. DESIGN: We examined anti-TRIB2 autoantibodies against a full-length [35S]-labeled TRIB2 antigen in Japanese patients with narcolepsy-cataplexy (n = 88), narcolepsy without cataplexy (n = 18), and idiopathic hypersomnia with long sleep time (n = 11). The results were compared to Japanese healthy controls (n = 87). Thirty-seven healthy control subjects were positive for HLA-DRB1*1501-DQB1*0602. We also examined autoantibodies against another Tribbles homolog, TRIB3, as an experimental control. MEASUREMENTS AND RESULTS: Autoantibodies against TRIB2 were found in 26.1% of patients with narcolepsy-cataplexy, a significantly higher prevalence than the 2.3% in healthy controls. We found that anti-TRIB3 autoantibodies were rare in patients with narcolepsy and showed no association with anti-TRIB2 indices. No significant correlation was found between anti-TRIB2 positivity and clinical information. CONCLUSIONS: We confirmed the higher prevalence and specificity of anti-TRIB2 autoantibodies in Japanese patients with narcolepsy-cataplexy. This suggests a subgroup within narcolepsy-cataplexy might be affected by an anti-TRIB2 autoantibody-mediated autoimmune mechanism.