Emmanuel Mignot

The Sleep Disorder Canine Narcolepsy Is Caused by a Mutation in the Hypocretin (Orexin) Receptor 2 Gene

Narcolepsy is a disabling sleep disorder affecting humans and animals. It is characterized by daytime sleepiness, cataplexy, and striking transitions from wakefulness into rapid eye movement (REM) sleep. In this study, we used positional cloning to identify an autosomal recessive mutation responsible for this sleep disorder in a well-established canine model. We have determined that canine narcolepsy is caused by disruption of the hypocretin (orexin) receptor 2 gene (Hcrtr2). This result identifies hypocretins as major sleep-modulating neurotransmitters and opens novel potential therapeutic approaches for narcoleptic patients.

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.

Short Sleep Duration Is Associated with Reduced Leptin, Elevated Ghrelin, and Increased Body Mass Index

Background Sleep duration may be an important regulator of body weight and metabolism. An association between short habitual sleep time and increased body mass index (BMI) has been reported in large population samples. The potential role of metabolic hormones in this association is unknown. Methods and Findings Study participants were 1,024 volunteers from the Wisconsin Sleep Cohort Study, a population-based longitudinal study of sleep disorders. Participants underwent nocturnal polysomnography and reported on their sleep habits through questionnaires and sleep diaries. Following polysomnography, morning, fasted blood samples were evaluated for serum leptin and ghrelin (two key opposing hormones in appetite regulation), adiponectin, insulin, glucose, and lipid profile. Relationships among these measures, BMI, and sleep duration (habitual and immediately prior to blood sampling) were examined using multiple variable regressions with control for confounding factors. A U-shaped curvilinear association between sleep duration and BMI was observed. In persons sleeping less than 8 h (74.4% of the sample), increased BMI was proportional to decreased sleep. Short sleep was associated with low leptin (p for slope = 0.01), with a predicted 15.5% lower leptin for habitual sleep of 5 h versus 8 h, and high ghrelin (p for slope = 0.008), with a predicted 14.9% higher ghrelin for nocturnal (polysomnographic) sleep of 5 h versus 8 h, independent of BMI. Conclusion Participants with short sleep had reduced leptin and elevated ghrelin. These differences in leptin and ghrelin are likely to increase appetite, possibly explaining the increased BMI observed with short sleep duration. In Western societies, where chronic sleep restriction is common and food is widely available, changes in appetite regulatory hormones with sleep curtailment may contribute to obesity.

Genetic and familial aspects of narcolepsy

Narcolepsy-cataplexy is a disabling sleep disorder characterized by excessive daytime sleepiness and abnormal REM sleep. The development of human narcolepsy involves environmental factors acting on a specific genetic background. The importance of environmental factors is evidenced by the reported 25 to 31% of monozygotic twins who are concordant for narcolepsy. One of the predisposing genetic factors is located in the MHC DQ region. More than 85% of all narcoleptic patients with definite cataplexy share a specific HLA allele, HLA DQB1*0602 (most often in combination with HLA DR2), compared with 12 to 38% of the general population, as evaluated in various ethnic groups. Genetic factors other than HLA are also likely to be involved. Even if genuine multiplex families are rare, 1 to 2% of the first-degree relatives of narcolepsy patients manifest the disorder, compared with 0.02 to 0.18% in the general population. Studies using a canine model of narcolepsy illustrate the importance of non-MHC genes in disease predisposition. In this model, narcolepsy is transmitted as a single autosomal recessive trait, canarc-1. In spite of an association with immune-related polymorphisms, narcolepsy does not appear to be a classic autoimmune disease. Other pathophysiologic models involving the microglia and the release of specific cytokines in the CNS may be involved and are being explored. This approach, together with positional cloning studies in humans and canines, should reveal the cause of narcolepsy and open new therapeutic avenues.

Complex HLA-DR and -DQ Interactions Confer Risk of Narcolepsy- Cataplexy in Three Ethnic Groups

Human narcolepsy-cataplexy, a sleep disorder associated with a centrally mediated hypocretin (orexin) deficiency, is tightly associated with HLA-DQB1*0602. Few studies have investigated the influence that additional HLA class II alleles have on susceptibility to this disease. In this work, 1,087 control subjects and 420 narcoleptic subjects with cataplexy, from three ethnic groups, were HLA typed, and the effects of HLA-DRB1, -DQA1, and -DQB1 were analyzed. As reported elsewhere, almost all narcoleptic subjects were positive for both HLA-DQA1*0102 and -DQB1*0602. A strong predisposing effect was observed in DQB1*0602 homozygotes, across all ethnic groups. Relative risks for narcolepsy were next calculated for heterozygous DQB1*0602/other HLA class II allelic combinations. Nine HLA class II alleles carried in trans with DQB1*0602 were found to influence disease predisposition. Significantly higher relative risks were observed for heterozygote combinations including DQB1*0301, DQA1*06, DRB1*04, DRB1*08, DRB1*11, and DRB1*12. Three alleles-DQB1*0601, DQB1*0501, and DQA1*01 (non-DQA1*0102)-were found to be protective. The genetic contribution of HLA-DQ to narcolepsy susceptibility was also estimated by use of lambda statistics. Results indicate that complex HLA-DR and -DQ interactions contribute to the genetic predisposition to human narcolepsy but that additional susceptibility loci are also most likely involved. Together with the recent hypocretin discoveries, these findings are consistent with an immunologically mediated destruction of hypocretin-containing cells in human narcolepsy-cataplexy.

Narcolepsy with cataplexy

Narcolepsy with cataplexy is a disabling sleep disorder affecting 0.02% of adults worldwide. It is characterised by severe, irresistible daytime sleepiness and sudden loss of muscle tone (cataplexy), and can be associated with sleep-onset or sleep-offset paralysis and hallucinations, frequent movement and awakening during sleep, and weight gain. Sleep monitoring during night and day shows rapid sleep onset and abnormal, shortened rapid-eye-movement sleep latencies. The onset of narcolepsy with cataplexy is usually during teenage and young adulthood and persists throughout the lifetime. Pathophysiological studies have shown that the disease is caused by the early loss of neurons in the hypothalamus that produce hypocretin, a wakefulness-associated neurotransmitter present in cerebrospinal fluid. The cause of neural loss could be autoimmune since most patients have the HLA DQB1*0602 allele that predisposes individuals to the disorder. Treatment is with stimulant drugs to suppress daytime sleepiness, antidepressants for cataplexy, and gamma hydroxybutyrate for both symptoms. Because narcolepsy is an under-recognised disease, it is important that general practitioners and other primary health-care workers identify abnormal daytime sleepiness early.

Pharmacological aspects of human and canine narcolepsy.

Narcolepsy-cataplexy is a disabling neurological disorder that affects 1/2000 individuals. The main clinical features of narcolepsy, excessive daytime sleepiness and symptoms of abnormal REM sleep (cataplexy, sleep paralysis, hypnagogic hallucinations) are currently treated using amphetamine-like compounds or modafinil and antidepressants. Pharmacological research in the area is facilitated greatly by the existence of a canine model of the disorder. The mode of action of these compounds involves presynaptic activation of adrenergic transmission for the anticataplectic effects of antidepressant compounds and presynaptic activation of dopaminergic transmission for the EEG arousal effects of amphetamine-like stimulants. The mode of action of modafmil is still uncertain, and other neurochemical systems may offer interesting avenues for therapeutic development. Pharmacological and physiological studies using the canine model have identified primary neurochemical and neuroanatomical systems that underlie the expression of abnormal REM sleep and excessive sleepiness in narcolepsy. These involve mostly the pontine and basal forebrain cholinergic, the pontine adrenergic and the mesolimbic and mesocortical dopaminergic systems. These studies confirm a continuing need for basic research in both human and canine narcolepsy, and new treatments that act directly at the level of the primary defect in narcolepsy might be forthcoming.

Fluctuation of extracellular hypocretin-1 (orexin A) levels in the rat in relation to the light-dark cycle and sleep-wake activities

Hypocretins/orexins are neuropeptides implicated in sleep regulation and the sleep disorder narcolepsy. In order to examine how hypocretin activity fluctuates across 24 h with respect to the sleep–wake cycle, we measured changes in extracellular hypocretin‐1 levels in the lateral hypothalamus and medial thalamus of freely moving rats with simultaneous sleep recordings. Hypocretin levels exhibited a robust diurnal fluctuation; levels slowly increased during the dark period (active phase), and decreased during the light period (rest phase). Levels were not correlated with the amount of wake or sleep in each period. Although an acute 4‐h light‐shift did not alter hypocretin levels, 6‐h sleep deprivation significantly increased hypocretin release during the forced‐wake period. Hypocretin activity is, thus, likely to build up during wakefulness and decline with the occurrence of sleep. These findings, together with the fact that a difficulty in maintaining wakefulness during the daytime is one of the primary symptoms of hypocretin‐deficient narcolepsy, suggest that hypocretin activity may be critical in opposing sleep propensity during periods of prolonged wakefulness.

CSF hypocretin/orexin levels in narcolepsy and other neurological conditions

Objective: To examine the specificity of low CSF hypocretin-1 levels in narcolepsy and explore the potential role of hypocretins in other neurologic disorders. Methods: A method to measure hypocretin-1 in 100 μL of crude CSF sample was established and validated. CSF hypocretin-1 was measured in 42 narcolepsy patients (ages 16–70 years), 48 healthy controls (ages 22–77 years,) and 235 patients with various other neurologic conditions (ages 0–85 years). Results: As previously reported, CSF hypocretin-1 levels were undetectably low (<100 pg/mL) in 37 of 42 narcolepsy subjects. Hypocretin-1 levels were detectable in all controls (224–653 pg/mL) and all neurologic patients (117–720 pg/mL), with the exception of three patients with Guillain–Barré syndrome (GBS). Hypocretin-1 was within the control range in most neurologic patients tested, including patients with AD, PD, and MS. Low but detectable levels (100–194 pg/mL) were found in a subset of patients with acute lymphocytic leukemia, intracranial tumors, craniocerebral trauma, CNS infections, and GBS. Conclusions: Undetectable CSF hypocretin-1 levels are highly specific to narcolepsy and rare cases of GBS. Measuring hypocretin-1 levels in the CSF of patients suspected of narcolepsy is a useful diagnostic procedure. Low hypocretin levels are also observed in a large range of neurologic conditions, most strikingly in subjects with head trauma. These alterations may reflect focal lesions in the hypothalamus, destruction of the blood brain barrier, or transient or chronic hypofunction of the hypothalamus. Future research in this area is needed to establish functional significance.

Narcolepsy is strongly associated with the T-cell receptor alpha locus

Narcolepsy with cataplexy, characterized by sleepiness and rapid onset into REM sleep, affects 1 in 2,000 individuals. Narcolepsy was first shown to be tightly associated with HLA-DR2 (ref. 3) and later sublocalized to DQB1*0602 (ref. 4). Following studies in dogs and mice, a 95% loss of hypocretin-producing cells in postmortem hypothalami from narcoleptic individuals was reported. Using genome-wide association (GWA) in Caucasians with replication in three ethnic groups, we found association between narcolepsy and polymorphisms in the TRA@ (T-cell receptor alpha) locus, with highest significance at rs1154155 (average allelic odds ratio 1.69, genotypic odds ratios 1.94 and 2.55, P < 10−21, 1,830 cases, 2,164 controls). This is the first documented genetic involvement of the TRA@ locus, encoding the major receptor for HLA-peptide presentation, in any disease. It is still unclear how specific HLA alleles confer susceptibility to over 100 HLA-associated disorders; thus, narcolepsy will provide new insights on how HLA–TCR interactions contribute to organ-specific autoimmune targeting and may serve as a model for over 100 other HLA-associated disorders.