Emergency drug kits at the Danish hospital pharmacies: varying management and challenges.

OBJECTIVES: Access to emergency drug kits (EDK) during medical emergencies can be life-saving; however, recent doubts about the quality of the kits have been expressed. Procurements of pharmaceuticals to the five regional authorities in Denmark are serviced by Amgros, a public sector organisation owned by the regions and established to create economies of scale and achieve administrative savings by centralisation. This means that Amgros calls for tenders for the supply of pharmaceuticals to the hospital pharmacies. The Hospital Pharmacy in the North Denmark Region does not currently have an effective method to manage Amgros procurements in relation to EDKs. Thus, the objectives were to explore how quality in the management and packing of EDKs is assured and maintained at different hospital pharmacies in Denmark and how this is affected by Amgros procurements. METHODS: The hospital pharmacies in Denmark were enrolled in a cross-sectional study. Information about the management and challenges of the EDKs was inquired by means of a questionnaire. Responses were analysed by simple statistics. RESULTS: All eight hospital pharmacies in Denmark completed the questionnaire, and the distribution between single-use and reusable packaging was nearly equal. The hospital pharmacies comply with a variation of regulations of which good distribution practice is the most common. Six hospital pharmacies experience challenges with drug replacements in the EDKs and only one hospital pharmacy complies completely with the Amgros procurement. The majority of the hospital pharmacies use parameters such as price of the new drug and potential expense for new packaging in their decision of whether to comply with the Amgros procurement. CONCLUSION: The management of the EDKs varies greatly among the hospital pharmacies in Denmark, and national requirements are therefore encouraged to ensure the quality. The challenges experienced with drug replacements reflect that complying with the Amgros procurement can be troublesome.

Hypocretin-deficient narcolepsy patients have abnormal brain activation during humor processing.

STUDY OBJECTIVES: To assess brain activation patterns in response to fun-rated and neutral-rated movies we performed functional magnetic resonance imaging (fMRI) during a humor-paradigm in narcolepsy type 1 (NT1) patients with cataplexy (muscle atonia triggered by emotions) and controls. METHODS: The fMRI-humor-paradigm consisted of short movies (25/30 with a humorous punchline; 5/30 without a humorous punchline [but with similar build-up/anticipation]) rated by participants based on their humor experience. We included 41 NT1 patients and 44 controls. Group-level inferences were made using permutation testing. RESULTS: Permutation testing revealed no group differences in average movie ratings. fMRI analysis found no group differences in brain activations to fun-rated movies. Patients showed significantly higher activations compared to controls during neutral-rated movies; including bilaterally in the thalamus, pallidum, putamen, amygdala, hippocampus, middle temporal gyrus, cerebellum, brainstem and in the left precuneus, supramarginal gyrus, and caudate. We found no brain overactivation for patients during movies without a humorous punchline (89.0% neutral-rated). Group analyses revealed significantly stronger differentiation between fun-rated and neutral-rated movies in controls compared with patients (patients showed no significant differentiation), including bilaterally in the inferior frontal gyrus, thalamus, putamen, precentral gyrus, lingual gyrus, supramarginal gyrus, occipital areas, temporal areas, cerebellum and in the right hippocampus, postcentral gyrus, pallidum, and insula. CONCLUSION: Patients showed significantly higher activations in several cortical and subcortical regions during neutral-rated movies, with no differentiation from activations during fun-rated movies. This lower threshold for activating the humor response (even during neutral-rated movies), might represent insight into the mechanisms associated with cataplexy.

CD8+ T cells from patients with narcolepsy and healthy controls recognize hypocretin neuron-specific antigens.

Narcolepsy Type 1 (NT1) is a neurological sleep disorder, characterized by the loss of hypocretin/orexin signaling in the brain. Genetic, epidemiological and experimental data support the hypothesis that NT1 is a T-cell-mediated autoimmune disease targeting the hypocretin producing neurons. While autoreactive CD4+ T cells have been detected in patients, CD8+ T cells have only been examined to a minor extent. Here we detect CD8+ T cells specific toward narcolepsy-relevant peptides presented primarily by NT1-associated HLA types in the blood of 20 patients with NT1 as well as in 52 healthy controls, using peptide-MHC-I multimers labeled with DNA barcodes. In healthy controls carrying the disease-predisposing HLA-DQB1*06:02 allele, the frequency of autoreactive CD8+ T cells was lower as compared with both NT1 patients and HLA-DQB1*06:02-negative healthy individuals. These findings suggest that a certain level of CD8+ T-cell reactivity combined with HLA-DQB1*06:02 expression is important for NT1 development.

Psychiatric symptoms in patients with post-H1N1 narcolepsy type 1 in Norway.

STUDY OBJECTIVES: Several studies have reported psychiatric comorbidity in patients with narcolepsy type 1 (NC1). The primary aim of this study was to explore the extent of psychiatric symptoms in a cohort of Norwegian NC1 patients, most of whom were H1N1-vaccinated. We also wanted to explore possible causes of the psychiatric symptoms seen in NC1. METHODS: Cross-sectional study. Psychiatric symptoms were assessed by the Achenbach System of Empirically Based Assessment (ASEBA) Child Behavior Check List (CBCL) in children and by Adult Self Report (ASR) in adults. RESULTS: The mean (SD) total T-scores were 58.6 (9.2) for children and 57.0 (9.8) for adults, these being mainly driven by internalizing problems. Internalizing symptom T-scores showed that 37.5% of the children and 33.3% of the adults were in the clinical range of concern. T-scores were lower when the questionnaire's sleep-related items were excluded. However, 27.5% of children and 22.2% of adults still remained within the total psychiatric symptoms clinical range. Psychiatric symptoms and excessive daytime sleepiness were not associated. However, in children fragmented sleep, measured by sleep-stage shift index was significantly negatively associated with all the psychiatric summary scores (all p ≤ 0.020), and awakening index was negatively associated with externalizing (p = 0.042) and total summary scores (p = 0.042). In adults, awakening index, but not sleep-stage shift index, was positively associated with internalizing score (p = 0.015). Hypocretin-1 levels showed no association with psychiatric symptoms. CONCLUSIONS: We found a high prevalence of psychiatric symptoms in NC1 patients. Fragmented sleep was significantly associated with psychiatric symptoms.

Changes in quality of life in individuals with narcolepsy type 1 after the H1N1-influenza epidemic and vaccination campaign in Norway: a two-year prospective cohort study.

OBJECTIVE: Cross-sectional studies show a lower health-related quality of life (HRQoL) in individuals with narcolepsy. We aimed to describe changes in HRQoL after two years of multidisciplinary follow-up in a cohort of mainly post-H1N1 vaccination narcolepsy type-1 (NT1) patients in Norway. METHODS: Prospective-cohort study. Narcolepsy diagnosis was based on the International Classification of Sleep Disorders (third edition). Psychiatric comorbidity was assessed using the Achenbach System of Empirically Based Assessment (ASEBA). HRQoL was evaluated with the Pediatric Quality of Life Inventory (PedsQL™ Generic Core Scales 4.0) at baseline and follow-up. Mean follow-up time was 20.7 (2.7) months. RESULTS: Thirty one patients (18 females) with NT1, mean age 14.6 (SD = 4.8) years answered questionnaires at baseline and follow-up. On a group level, the PedsQL Total Health Summary score significantly improved by a mean of 5.9 (95%CI = 0.4, 11.9), p = 0.038; this was mainly driven by improvements in the Physical Health Summary score by 9.8 (3.0, 16.5) points, p = 0.006 and the School Functioning Scale score by 7.5 (1.0, 13.9) points p = 0.025. The Total ASEBA score was correlated with PedsQL Total Health Summary score at baseline, but not with changes in HRQoL. Sodium oxybate (Xyrem®) treatment at follow up was positively associated with changes in PedsQL Total Health Summary score, after adjusting for age and gender, p = 0.027. CONCLUSION: HRQoL in NT1 patients improved after two years of follow-up. The use of sodium oxybate (Xyrem®) at follow-up was associated with increases in HRQoL. Psychiatric comorbidity was correlated with HRQoL at baseline but did not predict changes in HRQoL at follow-up.

Comparison of subjective and objective measures of constipation - Employing a new method for categorizing gastrointestinal symptoms.

INTRODUCTION: Correlations between subjective and objective measures of constipation have seldom been demonstrated. This could be due to multiple confounding factors in clinical studies and the broad span of symptoms represented in questionnaires used to assess constipation. We developed a new method for categorizing gastrointestinal (GI) symptoms into relevant symptom groups, and used this in a controlled experimental study aimed to investigate whether GI transit times and colonic volumes were correlated to self-reported GI symptoms. METHODS: Twenty-five healthy male participants were enrolled in a randomized, double-blinded, placebo-controlled, five-day crossover study with the treatments oxycodone and placebo. Objective measures of GI transit times and colonic volumes were obtained by the means of the 3D-Transit System and magnetic resonance colonography, whereas subjective GI symptoms were measures via three validated questionnaires. The symptoms were then categorized into five groups; "abdominal symptoms", "defecation difficulties", "incomplete bowel evacuation", "reduced bowel movement frequency", and "stool symptoms". Spearman's rank order correlation was used to determine correlations between the five groups of symptoms and the objective measures. RESULTS: No correlations between the GI symptoms and transit times or colonic volumes were found (all P > 0.05). DISCUSSION: GI transit times and colonic volumes were not correlated to self-reported GI symptoms even in a controlled experimental study and when symptoms were categorized into relevant symptom groups. Thus, both subjective and objective measures must be considered relevant when assessing constipation in clinical and research settings, ensuring that both physiological aspects as well as the severity and impact of symptoms experienced by patients can be assessed.

Widespread white matter changes in post-H1N1 patients with narcolepsy type 1 and first-degree relatives.

Study Objectives: To assess white matter involvement in H1N1-vaccinated hypocretin deficient patients with narcolepsy type 1 (NT1) compared with first-degree relatives (a potential risk group) and healthy controls. Methods: We compared four diffusion tensor imaging-based microstructural indices (fractional anisotropy [FA], mean diffusivity [MD], radial diffusivity [RD], and axial diffusivity [AD]) in 57 patients with NT1 (39 females, mean age 21.8 years, 51/57 H1N1-vaccinated, 57/57 HLA-DQB1*06:02-positive, 54/54 hypocretin-deficient), 54 first-degree relatives (29 females, mean age 19.1 years, 37/54 H1N1-vaccinated, 32/54 HLA-DQB1*06:02-positive), and 55 healthy controls (38 females, mean age 22.3 years). We tested for differences between these groups, for parametric effects (controls > first-degree relatives > patients) and associations in patients (cerebrospinal fluid [CSF] hypocretin-1 and disease duration) and first-degree relatives (HLA-DQB1*06:02 and H1N1-vaccination). We employed tract-based spatial statistics and used permutation testing and threshold-free cluster enhancement for inference. Results: Patients with NT1 had a widespread, bilateral pattern of significantly lower FA compared with first-degree relatives and healthy controls. Additionally, patients with NT1 also exhibited significantly higher RD and lower AD in several focal white matter clusters. The parametric model showed that first-degree relatives had intermediate values. Full sample of patients with NT1 showed no significant associations with disease duration or CSF hypocretin-1. Conclusions: Our study suggests widespread abnormal white matter involvement far beyond the already known focal hypothalamic pathology in NT1, possibly reflecting the combined effects of the loss of the widely projecting hypothalamic hypocretin neurons, and/or secondary effects of wake/sleep dysregulation. These findings demonstrate the importance of white matter pathology in NT1.

Absence of autoreactive CD4+ T-cells targeting HLA-DQA1*01:02/DQB1*06:02 restricted hypocretin/orexin epitopes in narcolepsy type 1 when detected by EliSpot.

Narcolepsy type 1, a neurological sleep disorder strongly associated with Human Leukocyte Antigen (HLA-)DQB1*06:02, is caused by the loss of hypothalamic neurons producing the wake-promoting neuropeptide hypocretin (hcrt, also known as orexin). This loss is believed to be caused by an autoimmune reaction. To test whether hcrt itself could be a possible target in the autoimmune attack, CD4+ T-cell reactivity towards six different 15-mer peptides from prepro-hypocretin with high predicted affinity to the DQA1*01:02/DQB1*06:02 MHC class II dimer was tested using EliSpot in a cohort of 22 narcolepsy patients with low CSF hcrt levels, and 23 DQB1*06:02 positive healthy controls. Our ELISpot assay had a detection limit of 1:10,000 cells. We present data showing that autoreactive CD4+ T-cells targeting epitopes from the hcrt precursor in the context of MHC-DQA1*01:02/DQB1*06:02 are either not present or present in a frequency is <1:10,000 among peripheral CD4+ T-cells from narcolepsy type 1 patients.

Narcolepsy.

Narcolepsy is a chronic sleep disorder that has a typical onset in adolescence and is characterized by excessive daytime sleepiness, which can have severe consequences for the patient. Problems faced by patients with narcolepsy include social stigma associated with this disease, difficulties in obtaining an education and keeping a job, a reduced quality of life and socioeconomic consequences. Two subtypes of narcolepsy have been described (narcolepsy type 1 and narcolepsy type 2), both of which have similar clinical profiles, except for the presence of cataplexy, which occurs only in patients with narcolepsy type 1. The pathogenesis of narcolepsy type 1 is hypothesized to be the autoimmune destruction of the hypocretin-producing neurons in the hypothalamus; this hypothesis is supported by immune-related genetic and environmental factors associated with the disease. However, direct evidence in support of the autoimmune hypothesis is currently unavailable. Diagnosis of narcolepsy encompasses clinical, electrophysiological and biological evaluations, but simpler and faster procedures are needed. Several medications are available for the symptomatic treatment of narcolepsy, all of which have quite good efficacy and safety profiles. However, to date, no treatment hinders or slows disease development. Improved diagnostic tools and increased understanding of the pathogenesis of narcolepsy type 1 are needed and might lead to therapeutic or even preventative interventions.

Normal Morning Melanin-Concentrating Hormone Levels and No Association with Rapid Eye Movement or Non-Rapid Eye Movement Sleep Parameters in Narcolepsy Type 1 and Type 2.

STUDY OBJECTIVES: Other than hypocretin-1 (HCRT-1) deficiency in narcolepsy type 1 (NT1), the neurochemical imbalance of NT1 and narcolepsy type 2 (NT2) with normal HCRT-1 levels is largely unknown. The neuropeptide melanin-concentrating hormone (MCH) is mainly secreted during sleep and is involved in rapid eye movement (REM) and non-rapid eye movement (NREM) sleep regulation. Hypocretin neurons reciprocally interact with MCH neurons. We hypothesized that altered MCH secretion contributes to the symptoms and sleep abnormalities of narcolepsy and that this is reflected in morning cerebrospinal fluid (CSF) MCH levels, in contrast to previously reported normal evening/afternoon levels. METHODS: Lumbar CSF and plasma were collected from 07:00 to 10:00 from 57 patients with narcolepsy (subtypes: 47 NT1; 10 NT2) diagnosed according to International Classification of Sleep Disorders, Third Edition (ICSD-3) and 20 healthy controls. HCRT-1 and MCH levels were quantified by radioimmunoassay and correlated with clinical symptoms, polysomnography (PSG), and Multiple Sleep Latency Test (MSLT) parameters. RESULTS: CSF and plasma MCH levels were not significantly different between narcolepsy patients regardless of ICSD-3 subtype, HCRT-1 levels, or compared to controls. CSF MCH and HCRT-1 levels were not significantly correlated. Multivariate regression models of CSF MCH levels, age, sex, and body mass index predicting clinical, PSG, and MSLT parameters did not reveal any significant associations to CSF MCH levels. CONCLUSIONS: Our study shows that MCH levels in CSF collected in the morning are normal in narcolepsy and not associated with the clinical symptoms, REM sleep abnormalities, nor number of muscle movements during REM or NREM sleep of the patients. We conclude that morning lumbar CSF MCH measurement is not an informative diagnostic marker for narcolepsy.

Cerebrospinal fluid cytokine levels in type 1 narcolepsy patients very close to onset.

Type 1 narcolepsy is caused by a loss of hypocretin (orexin) signaling in the brain. Genetic data suggests the disorder is caused by an autoimmune attack on hypocretin producing neurons in hypothalamus. This hypothesis has however not yet been confirmed by consistent findings of autoreactive antibodies or T-cells in patient samples. One explanation for these negative results may be that the autoimmune process is no longer active when patients present to the clinic. With increasing awareness in recent years, more and more patients have been diagnosed closer and closer to disease onset. In this study, we tested whether an active immune process in the brain could be detected in these patients, as reflected by increased cytokine levels in the cerebrospinal fluid (CSF). Using multiplex analysis, we measured the levels of 51 cytokines and chemokines in the CSF of 40 type 1 narcolepsy patients having varying disease duration. For comparison, we used samples from 9 healthy controls and 9 patients with other central hypersomnia. Cytokine levels did not differ significantly between controls and patients, even in 5 patients with disease onset less than a month prior to CSF sampling.

HLA-DPB1 and HLA class I confer risk of and protection from narcolepsy.

Type 1 narcolepsy, a disorder caused by a lack of hypocretin (orexin), is so strongly associated with human leukocyte antigen (HLA) class II HLA-DQA1(∗)01:02-DQB1(∗)06:02 (DQ0602) that very few non-DQ0602 cases have been reported. A known triggering factor for narcolepsy is pandemic 2009 influenza H1N1, suggesting autoimmunity triggered by upper-airway infections. Additional effects of other HLA-DQ alleles have been reported consistently across multiple ethnic groups. Using over 3,000 case and 10,000 control individuals of European and Chinese background, we examined the effects of other HLA loci. After careful matching of HLA-DR and HLA-DQ in case and control individuals, we found strong protective effects of HLA-DPA1(∗)01:03-DPB1(∗)04:02 (DP0402; odds ratio [OR] = 0.51 [0.38-0.67], p = 1.01 × 10(-6)) and HLA-DPA1(∗)01:03-DPB1(∗)04:01 (DP0401; OR = 0.61 [0.47-0.80], p = 2.07 × 10(-4)) and predisposing effects of HLA-DPB1(∗)05:01 in Asians (OR = 1.76 [1.34-2.31], p = 4.71 × 10(-05)). Similar effects were found by conditional analysis controlling for HLA-DR and HLA-DQ with DP0402 (OR = 0.45 [0.38-0.55] p = 8.99 × 10(-17)) and DP0501 (OR = 1.38 [1.18-1.61], p = 7.11 × 10(-5)). HLA-class-II-independent associations with HLA-A(∗)11:01 (OR = 1.32 [1.13-1.54], p = 4.92 × 10(-4)), HLA-B(∗)35:03 (OR = 1.96 [1.41-2.70], p = 5.14 × 10(-5)), and HLA-B(∗)51:01 (OR = 1.49 [1.25-1.78], p = 1.09 × 10(-5)) were also seen across ethnic groups in the HLA class I region. These effects might reflect modulation of autoimmunity or indirect effects of HLA class I and HLA-DP alleles on response to viral infections such as that of influenza.

Reduced CSF hypocretin-1 levels are associated with cluster headache.

BACKGROUND: Cluster headache (CH) is a debilitating disorder characterized by unilateral, severe pain attacks with accompanying autonomic symptoms, often waking the patient from sleep. As it exhibits strong chronobiological traits and genetic studies have suggested a link with the hypocretin (HCRT) system, the objective of this study was to investigate HCRT-1 in CH patients. METHODS: Cerebrospinal fluid HCRT-1 concentration was measured in 12 chronic and 14 episodic CH patients during an active bout, and in 27 healthy controls. The patients were well characterized and clinical features compared to the HCRT concentration. RESULTS: We found significantly lower HCRT levels both in chronic (p = 0.0221) and episodic CH (p = 0.0005) patients compared with controls. No significant relationship was found with other clinical features. CONCLUSIONS: This is the first report of significantly reduced HCRT concentrations in CH patients. We speculate that decreased HCRT may reflect insufficient antinociceptive activity of the hypothalamus. The mechanism of the antinociceptive effect of HCRT is not known and requires further investigation. This study supports the hypothesis of a connection between arousal regulation and CH.

MiRNA profiles in cerebrospinal fluid from patients with central hypersomnias.

MicroRNAs (miRNAs) are involved in the pathogenesis of many human diseases, including some neurological disorders. Recently, we have reported dysregulated miRNAs in plasma from patients with central hypersomnias including type 1 and type 2 narcolepsy, and idiopathic hypersomnia. This study addressed whether miRNA levels are altered in the cerebrospinal fluid (CSF) of patients with central hypersomnias. We conducted high-throughput analyses of miRNAs in CSF from patients using quantitative real-time polymerase chain reaction panels. We identified 13, 9, and 11 miRNAs with a more than two-fold change in concentration in CSF from patients with type 1 and type 2 narcolepsy and idiopathic hypersomnia, respectively, compared with matched healthy controls. Most miRNAs differed in more than one of the sleep disorders. However, all miRNAs were detected at low levels in CSF and varied between individuals. None of them showed significant differences in concentrations between groups after correcting for multiple testing, and none could be validated in an independent cohort. Nevertheless, approximately 60% of the most abundant miRNAs in the profile reported here have previously been identified in the CSF of healthy individuals, showing consistency with previous miRNA profiles found in CSF. In conclusion, we were not able to demonstrate distinct levels or patterns of miRNAs in CSF from central hypersomnia patients.

Sleep-wake transition in narcolepsy and healthy controls using a support vector machine.

Narcolepsy is characterized by abnormal sleep-wake regulation, causing sleep episodes during the day and nocturnal sleep disruptions. The transitions between sleep and wakefulness can be identified by manual scorings of a polysomnographic recording. The aim of this study was to develop an automatic classifier capable of separating sleep epochs from epochs of wakefulness by using EEG measurements from one channel. Features from frequency bands α (0-4 Hz), ß (4-8 Hz), δ (8-12 Hz), θ (12-16 Hz), 16 to 24 Hz, 24 to 32 Hz, 32 to 40 Hz, and 40 to 48 Hz were extracted from data by use of a wavelet packet transformation and were given as input to a support vector machine classifier. The classification algorithm was assessed by hold-out validation and 10-fold cross-validation. The data used to validate the classifier were derived from polysomnographic recordings of 47 narcoleptic patients (33 with cataplexy and 14 without cataplexy) and 15 healthy controls. Compared with manual scorings, an accuracy of 90% was achieved in the hold-out validation, and the area under the receiver operating characteristic curve was 95%. Sensitivity and specificity were 90% and 88%, respectively. The 10-fold cross-validation procedure yielded an accuracy of 88%, an area under the receiver operating characteristic curve of 92%, a sensitivity of 87%, and a specificity of 87%. Narcolepsy with cataplexy patients experienced significantly more sleep-wake transitions during night than did narcolepsy without cataplexy patients (P = 0.0199) and healthy subjects (P = 0.0265). In addition, the sleep-wake transitions were elevated in hypocretin-deficient patients. It is concluded that the classifier shows high validity for identifying the sleep-wake transition. Narcolepsy with cataplexy patients have more sleep-wake transitions during night, suggesting instability in the sleep-wake regulatory system.

EGFR signaling patterns are regulated by its different ligands.

EGF receptor (EGFR) and its signaling have been investigated for many years, but how its different ligands regulate signaling has not been thoroughly explored. When investigating EGFR activation and downstream signaling in HeLa cells using a panel of ligands, we found a ligand-dependent differential activation of EGFR and the signaling pathways Akt, PLCγ and STAT with HB-EGF and BTC being the most potent ligands. All the tested ligands induced full activation of Erk signaling at 1 nM, whereas only HB-EGF and partly BTC and EGF induced strong activation of Akt, STAT3 and PLCγ at this concentration. Interestingly, we also found that the high activation potencies of HB-EGF and BTC could only partially be explained by their binding affinities, and are therefore likely to be regulated by other mechanisms. We thus suggest that the signaling pathways initiated from the EGFR vary depending on the ligands bound in a cell specific manner.

HLA DQB1*06:02 negative narcolepsy with hypocretin/orexin deficiency.

STUDY OBJECTIVES: To identify rare allelic variants and HLA alleles in narcolepsy patients with hypocretin (orexin, HCRT) deficiency but lacking DQB1*06:02. SETTINGS: China (Peking University People's Hospital), Czech Republic (Charles University), Denmark (Golstrup Hospital), Italy (University of Bologna), Korea (Catholic University), and USA (Stanford University). DESIGN: CSF hypocretin-1, DQB1*06:02, clinical and polysomnographic data were collected in narcolepsy patients (552 with and 144 without cataplexy) from 6 sites. Numbers of cases with and without DQB1*06:02 and low CSF hypocretin-1 were compiled. HLA class I (A, B, C), class II (DRBs, DQA1, DQB1, DPA1, and DPB1), and whole exome sequencing were conducted in 9 DQB1*06:02 negative cases with low CSF hypocretin-1. Sanger sequencing of selected exons in DNMT1, HCRT, and MOG was performed to exclude mutations in known narcolepsy-associated genes. MEASUREMENTS AND RESULTS: Classic narcolepsy markers DQB1*06:02 and low CSF hypocretin-1 were found in 87.4% of cases with cataplexy, and in 20.0% without cataplexy. Nine cases (all with cataplexy) were DQB1*06:02 negative with low CSF hypocretin-1, constituting 1.7% [0.8%-3.4%] of all cases with cataplexy and 1.8% [0.8%-3.4%] of cases with low CSF hypocretin independent of cataplexy across sites. Five HLA negative subjects had severe cataplexy, often occurring without clear triggers. Subjects had diverse ethnic backgrounds and HLA alleles at all loci, suggesting no single secondary HLA association. The rare subtype DPB1*0901, and homologous DPB1*10:01 subtype, were present in 5 subjects, suggesting a secondary association with HLA-DP. Preprohypocretin sequencing revealed no mutations beyond one previously reported in a very early onset case. No new MOG or DNMT1 mutations were found, nor were suspicious or private variants in novel genes identified through exome sequencing. CONCLUSIONS: Hypocretin, MOG, or DNMT1 mutations are exceptional findings in DQB1*06:02 negative cases with hypocretin deficiency. A secondary HLA-DP association may be present in these cases. These represent particularly difficult diagnostic challenges.