LC-MS/MS Quantitation of HILIC-Enriched N-glycopeptides Derived from Low-Abundance Serum Glycoproteins in Patients with Narcolepsy Type 1.

Glycoproteomic analysis is always challenging because of low abundance and complex site-specific heterogeneity. Glycoproteins are involved in various biological processes such as cell signaling, adhesion, and cell-cell communication and may serve as potential biomarkers when analyzing different diseases. Here, we investigate glycoproteins in narcolepsy type 1 (NT1) disease, a form of narcolepsy characterized by cataplexy-the sudden onset of muscle paralysis that is typically triggered by intense emotions. In this study, 27 human blood serum samples were analyzed, 16 from NT1 patients and 11 from healthy individuals serving as controls. We quantified hydrophilic interaction liquid chromatography (HILIC)-enriched glycopeptides from low-abundance serum samples of controls and NT1 patients via LC-MS/MS. Twenty-eight unique N-glycopeptides showed significant changes between the two studied groups. The sialylated N-glycopeptide structures LPTQNITFQTESSVAEQEAEFQSPK HexNAc6, Hex3, Neu5Ac2 (derived from the ITIH4 protein) and the structure IVLDPSGSMNIYLVLDGSDSIGASNFTGAK HexNAc5, Hex4, Fuc1 (derived from the CFB protein), with p values of 0.008 and 0.01, respectively, were elevated in NT1 samples compared with controls. In addition, the N-glycopeptide protein sources Ceruloplasmin, Complement factor B, and ITH4 were observed to play an important role in the complement activation and acute-phase response signaling pathways. This may explain the possible association between the biomarkers and pathophysiological effects.

Narcolepsy risk loci outline role of T cell autoimmunity and infectious triggers in narcolepsy.

Narcolepsy type 1 (NT1) is caused by a loss of hypocretin/orexin transmission. Risk factors include pandemic 2009 H1N1 influenza A infection and immunization with Pandemrix®. Here, we dissect disease mechanisms and interactions with environmental triggers in a multi-ethnic sample of 6,073 cases and 84,856 controls. We fine-mapped GWAS signals within HLA (DQ0602, DQB1*03:01 and DPB1*04:02) and discovered seven novel associations (CD207, NAB1, IKZF4-ERBB3, CTSC, DENND1B, SIRPG, PRF1). Significant signals at TRA and DQB1*06:02 loci were found in 245 vaccination-related cases, who also shared polygenic risk. T cell receptor associations in NT1 modulated TRAJ*24, TRAJ*28 and TRBV*4-2 chain-usage. Partitioned heritability and immune cell enrichment analyses found genetic signals to be driven by dendritic and helper T cells. Lastly comorbidity analysis using data from FinnGen, suggests shared effects between NT1 and other autoimmune diseases. NT1 genetic variants shape autoimmunity and response to environmental triggers, including influenza A infection and immunization with Pandemrix®.

LC-MS/MS-Based Proteomics Approach for the Identification of Candidate Serum Biomarkers in Patients with Narcolepsy Type 1.

Narcolepsy type 1 (NT1) is the most common type of narcolepsy known to be caused by the loss of specific neurons responsible for producing peptide neurotransmitters (orexins/hypocretins), resulting in a sleep-wake cycle disorder. It is characterized by its association with cataplexy and abnormalities in rapid eye movement. To date, no cure has been established for this life-threatening condition. Misdiagnosis of NT1 is also quite common, although it is not exceedingly rare. Therefore, successfully identifying candidate serum biomarkers for NT1 would be a head start for accurate diagnosis and development of therapeutics for this disorder. This study aims to identify such potential serum biomarkers. A depletion protocol was employed for 27 human serum samples (16 NT1 and 11 healthy controls), followed by applying LC-MS/MS bottom-up proteomics analysis, then LC-PRM-MS for validation. The comparison of the proteome profiles of the low-abundant proteins in the samples was then investigated based on age, sex, sample groups, and the presence of the Human Leukocyte Antigen (HLA) DQB1*0602 allele. The results were tracked to gene expression studies as well as system biology to identify key proteins and understand their relationship in the pathogenesis of NT1. Our results revealed 36 proteins significantly and differentially expressed. Among the impaired pathways and bioprocesses, the complement activation pathway is impaired by six of the differentially expressed proteins (DEPs). They are coded by the genes C2, CFB, C5, C1R, C1S, and MASP1, while 11 DEPs are involved in Acute Phase Response Signaling (APRS), which are coded by the genes FN1, AMBP, APOH, CFB, CP, ITIH2, C5, C2, F2, C1, and ITIH4. The combined AUCs of the downregulated and upregulated DEPs are 0.95 and 0.76, respectively. Overall, this study reveals potential serum-protein biomarkers of NT1 and explains the possible correlation between the biomarkers and pathophysiological effects, as well as important biochemical pathways involved in NT1.

Repeated measures of hypocretin-1 in Danish and Italian patients with narcolepsy and in controls.

STUDY OBJECTIVES: The assay currently used worldwide to measure cerebrospinal fluid hypocretin-1 (CSF-hcrt-1) for diagnosing narcolepsy uses a competitive radioimmunoassay with polyclonal anti-hcrt-1 antibodies. This assay detects multiple hypocretin-1 immunoreactive species in the CSF that are all derived from full-length hcrt-1. We aimed to revalidate CSF-hcrt-1 cut-offs for narcolepsy type 1 (NT1) diagnosis and to evaluate temporal changes in CSF-hcrt-1 levels in patients suspected of having central hypersomnia. METHOD: We carried out a repeat lumbar puncture with a mean follow-up of 4.0 years, to measure CSF-hcrt-1 in patients suspected of having central hypersomnia in a follow-up study. Data from CSF samples of patients with NT1 and of controls without known hypersomnia, from the Italian-Stanford and Danish populations, were examined using a receiver-operating characteristic analysis. RESULTS: The optimal CSF-hcrt-1 cut-offs for identifying NT1 were 129 pg/ml and 179 pg/ml for the Italian-Stanford and Danish populations, respectively. The sensitivity was 0.93-0.99 and the specificity was 1. Follow-up lumbar puncture measurements of CSF-hcrt-1 were obtained from 73 patients. 30 of 32 patients with low CSF-hcrt-1 levels continued to be categorized as low, with an unaltered diagnosis; two patients showed a marked increase in CSF-hcrt-1, attaining normal values at follow-up. One of these patients relapsed to low CSF-hcrt-1 after follow-up. All 41 patients with normal CSF-hcrt-1 at baseline had normal CSF-hcrt-1 at follow-up. CONCLUSION: CSF-hcrt-1 measurement can provide an accurate test for diagnosing NT1, although it is important to validate the CSF-hcrt-1 cut-off for specific testing locations. Stable CSF-hcrt-1 levels support the already established prognosis of narcolepsy as permanent once the disorder has fully developed.

Multidisciplinary care of patients with narcolepsy during coronavirus disease 2019 pandemic in Italy via televisit: the TElemedicine for NARcolepsy feasibility study.

STUDY OBJECTIVES: Narcolepsy is a rare chronic central disorder of hypersomnolence with frequent endocrine-metabolic comorbidities. To address the complex care needs of patients during the COVID-19 emergency, we carried out a feasibility study of the TElemedicine for NARcolepsy (TENAR) protocol with the aim of assessing the feasibility of a multidisciplinary care approach via televisit for patients with narcolepsy. METHODS: A feasibility single open-arm study on the multidisciplinary care of children (>7 y.o.) and adults with narcolepsy who required a follow-up visit was realized during the COVID-19 pandemic emergency period in Italy. The study included a sleep, metabolic, and psychosocial assessment via televisit at baseline, at 6, and at 12 months from the study inclusion period (15th May-26th June 2020). RESULTS: In total 39 out of 44 eligible patients (89%) entered the study (30 adults, nine children); 37 patients (95%) ended the 12-month follow-up. At baseline, the median Epworth sleepiness scale score (ESS) was 10 (IQR 8-14), and the median body mass index (BMI) was 25.6 (IQR 22.1-30.9). During the follow-up period, the ESS score decreased from the 6th month onward (p = 0.003), and BMI decreased at the 1-year follow-up (p = 0.047), while there were no differences in depressive and anxiety symptoms, quality of life, compliance with treatment, adverse drug reactions, or accidents. CONCLUSIONS: High response and retention rates, stability of ESS, and lack of side effects indicate that telemedicine is a feasible and safe approach for adults and children with narcolepsy.

The role of mtDNA haplogroups on metabolic features in narcolepsy type 1.

Narcolepsy type 1 (NT1) is due to selective loss of hypocretin (hcrt)-producing-neurons. Hcrt is a neuropeptide regulating the sleep/wake cycle, as well as feeding behavior. A subset of NT1 patients become overweight/obese, with a dysmetabolic phenotype. We hypothesized that mitochondrial DNA (mtDNA) sequence variation might contribute to the metabolic features in NT1 and we undertook an exploratory survey of mtDNA haplogroups in a cohort of well-characterized patients. We studied 246 NT1 Italian patients, fully defined for their metabolic features, including obesity, hypertension, low HDL, hypertriglyceridemia and hyperglycemia. For haplogroup assignment, the mtDNA control region was sequenced in combination with an assessment of diagnostic markers in the coding region. NT1 patients displayed the same mtDNA haplogroups (H, HV, J, K, T, U) frequency as those reported in the general Italian population. The majority of NT1 patients (64%) were overweight: amongst these, 35% were obese, 48% had low HDL cholesterol levels, and 31% had hypertriglyceridemia. We identified an association between haplogroups J, K and hypertriglyceridemia (P = 0.03, 61.5% and 61.5%, respectively vs. 31.3% of the whole sample) and after correction for age and sex, we observed a reduction of these associations (OR = 3.65, 95%CI = 0.76-17.5, p = 0.106 and 1.73, 0.52-5.69, p = 0.368, respectively). The low HDL level showed a trend for association with haplogroup J (P = 0.09, 83.3% vs. 47.4% of the whole sample) and after correction we observed an OR = 6.73, 95%CI = 0.65-69.9, p = 0.110. Our study provides the first indication that mtDNA haplogroups J and K can modulate metabolic features of NT1 patients, linking mtDNA variation to the dysmetabolic phenotype in NT1.

The importance of social zeitgeber in paediatric type 1 narcolepsy: What we can learn from the COVID-19 restrictions adopted in Italy?

The lockdown due to the new coronavirus pandemic (COVID-19) has led to unparalleled changes in several aspects of human behaviour. During the lockdown, the general population delayed sleep timing and spent more time in bed; however, little is known on the effects of COVID-19 restriction on children and adolescents suffering type 1 narcolepsy. In the last months of 2019, we performed follow-up actigraphy in 18 type 1 narcolepsy children and adolescents under stable pharmacological treatment with sodium oxybate. We contacted these patients for a follow-up actigraphy during the first Italian lockdown. Actigraphs and the Epworth Sleepiness Scale for children and adolescents (ESS-CHAD) have been sent to participants' homes. Differences in motor activity were analysed through functional linear modelling. During lockdown, type 1 narcolepsy children and adolescents went to bed and woke up later, slept more during the daytime and napped more frequently. No difference emerged in time in bed, estimated total sleep time and nocturnal sleep quality. Similarly, no difference emerged in ESS-CHAD and body mass index. The time-series analysis of motor activity documented reduced activity during the early morning and in the evening during the lockdown period compared with pre-lockdown. Our study objectively showed that type 1 narcolepsy children and adolescents delayed the sleep phase and slept more during the daytime during the lockdown. The analysis of type 1 narcolepsy children and adolescents' behaviour during the lockdown has provided new information that could pave the way to a personalized school programme.

Searching for Novel Candidate Biomarkers of RLS in Blood by Proteomic Analysis.

PURPOSE: We performed comparative proteomic analyses of blood of patients with RLS and healthy individuals aiming to identify potential biomarker and therapeutic target candidate for RLS. PATIENTS AND METHODS: Blood serum samples from 12 patients with a clinical diagnosis of RLS (8 females and 4 males, with a mean age of 68.52 years) and 10 healthy controls (5 females and 5 males, with a mean age of 67.61 years) underwent proteomic profiling by liquid chromatography coupled with tandem mass spectrometry. Pathway analysis incorporating protein-protein interaction networks was carried out to identify pathological processes linked to the differentially expressed proteins. RESULTS: We quantified 272 proteins in patients with RLS and healthy controls, of which 243 were shared. Five proteins - apolipoprotein C-II, leucine-rich alpha-2-glycoprotein 1, FLJ92374, extracellular matrix protein 1, and FLJ93143 - were substantially increased in RLS patients, whereas nine proteins - vitamin D-binding protein, FLJ78071, alpha-1-antitrypsin, CD5 antigen-like, haptoglobin, fibrinogen alpha chain, complement factor H-related protein 1, platelet factor 4, and plasma protease C1 inhibitor - were decreased. Bioinformatics analyses revealed that these proteins were linked to 1) inflammatory and immune response, and complement activation, 2) brain-related development, cell aging, and memory disorders, 3) pregnancy and associated complications, 4) myocardial infarction, and 5) reactive oxygen species generation and subsequent diabetes mellitus. CONCLUSION: Our findings shed light on the multifactorial nature of RLS and identified a set of circulating proteins that may have clinical importance as biomarkers and therapeutic targets.

Onset of narcolepsy type 1 in a paraneoplastic encephalitis associated with a thymic seminoma.

Narcolepsy type 1 results from probable autoimmune disruption of hypothalamic hypocretinergic neurons. Secondary narcolepsy can occur as a result of other conditions affecting the central nervous system, including limbic paraneoplastic encephalitis. We report the case of a 19-year-old patient presenting with acute-onset diurnal hypersomnolence, hyperphagia, sexual dysfunction, and psychiatric disturbances. Further investigations revealed a limbic paraneoplastic encephalitis associated with mediastinal thymic seminoma. Tumor removal and immunosuppressive treatment resulted in a partial benefit on psychiatric disturbances but did not improve daytime sleepiness. A comprehensive sleep assessment led to the diagnosis of secondary narcolepsy type 1 with reduced cerebrospinal fluid hypocretin-1 levels and revealed the presence of the HLA DQB1*0602 allele, typically associated with idiopathic narcolepsy, for which we hypothesize a possible immunopathogenic role. Sodium oxybate was successfully administered. Narcolepsy is often overlooked in patients with limbic paraneoplastic encephalitis. A prompt assessment and an adequate symptomatic treatment can improve the disease burden. CITATION: Rossi S, Asioli GM, Rizzo G, et al. Onset of narcolepsy type 1 in a paraneoplastic encephalitis associated with a thymic seminoma. J Clin Sleep Med. 2021;17(12):2557-2560.

Pre-treatment of blood samples reveal normal blood hypocretin/orexin signal in narcolepsy type 1.

The hypocretin/orexin system regulates arousal through central nervous system mechanisms and plays an important role in sleep, wakefulness and energy homeostasis. It is unclear whether hypocretin peptides are also present in blood due to difficulties in measuring reliable and reproducible levels of the peptides in blood samples. Lack of hypocretin signalling causes the sleep disorder narcolepsy type 1, and low concentration of cerebrospinal fluid hypocretin-1/orexin-A peptide is a hallmark of the disease. This measurement has high diagnostic value, but performing a lumbar puncture is not without discomfort and possible complications for the patient. A blood-based test to assess hypocretin-1 deficiency would therefore be of obvious benefit. We here demonstrate that heating plasma or serum samples to 65°C for 30 min at pH 8 significantly increases hypocretin-1 immunoreactivity enabling stable and reproducible measurement of hypocretin-1 in blood samples. Specificity of the signal was verified by high-performance liquid chromatography and by measuring blood samples from mice lacking hypocretin. Unspecific background signal in the assay was high. Using our method, we show that hypocretin-1 immunoreactivity in blood samples from narcolepsy type 1 patients does not differ from the levels detected in control samples. The data presented here suggest that hypocretin-1 is present in the blood stream in the low picograms per millilitres range and that peripheral hypocretin-1 concentrations are unchanged in narcolepsy type 1.

Impact of COVID-19 pandemic lockdown on narcolepsy type 1 management.

STUDY OBJECTIVES: Narcolepsy type 1 (NT1) is a chronic rare hypersomnia of central origin requiring a combination of behavioral and pharmacological treatments. During the coronavirus disease 2019 (COVID-19) pandemic, in Italy the population was forced into a lockdown. With this study, we aimed to describe the lockdown impact on NT1 symptom management, according to different patients' working schedule. METHODS: In the period between 10 April and 15 May 2020, we performed routine follow-up visits by telephone (as recommended during the COVID-19 emergency) to 50 patients >18 years old (40% males) under stable long-term treatment. We divided patients into three groups: unchanged working schedule, forced working/studying at home, and those who lost their job ("lost occupation"). Current sleep-wake habit and symptom severity were compared with prelockdown assessment (six months before) in the three patient groups. RESULTS: At assessment, 20, 22, and eight patients belonged to the unchanged, working/studying at home, and lost occupation groups, respectively. While in the lost occupation group, there were no significant differences compared with prepandemic assessment, the patients with unchanged schedules reported more nocturnal awakenings, and NT1 patients working/studying at home showed an extension of nocturnal sleep time, more frequent daytime napping, improvement of daytime sleepiness, and a significant increase in their body mass index. Sleep-related paralysis/hallucinations, automatic behaviors, cataplexy, and disturbed nocturnal sleep did not differ. CONCLUSIONS: Narcolepsy type 1 patients working/studying at home intensified behavioral interventions (increased nocturnal sleep time and daytime napping) and ameliorated daytime sleepiness despite presenting with a slight, but significant, increase of weight.

REM Sleep Behavior Disorder in Children With Type 1 Narcolepsy Treated With Sodium Oxybate.

OBJECTIVE: To study the effect of stable treatment with sodium oxybate (SO) on nocturnal REM sleep behavior disorder (RBD) and REM sleep without atonia (RSWA) that severely affected children with type 1 narcolepsy (NT1). METHODS: Nineteen children and adolescents with NT1 (9 female, mean age 12.5 ± 2.7 years, mean disease duration 3.4 ± 1.6 years) underwent neurologic investigations and video-polysomnography (v-PSG) at baseline and after 3 months of stable treatment with SO. v-PSG was independently analyzed by 2 sleep experts to rate RBD episodes. RSWA was automatically computed by means of the validated REM sleep atonia index (RAI). RESULTS: Compared to baseline, RAI significantly improved (p < 0.05) and complex movements during REM sleep were remarkably reduced after stable treatment with SO. Compared to baseline, children also reported improvement in clinical complaints and showed a different nighttime sleep-stage architecture. CONCLUSIONS: RBD and RSWA improved after treatment with SO, pointing to a direct role of the drug in modulating motor control during REM sleep. CLASSIFICATION OF EVIDENCE: This study offers Class IV evidence of the positive effect of SO on modulation of muscle atonia during REM sleep in children with NT1 because of the absence of a control group.

Protocols of a diagnostic study and a randomized controlled non-inferiority trial comparing televisits vs standard in-person outpatient visits for narcolepsy diagnosis and care: TElemedicine for NARcolepsy (TENAR).

BACKGROUND: Narcolepsy is a rare chronic sleep disorder that typically begins in youth. Excessive daytime sleepiness is the main disabling symptom, but the disease is often associated with severe endocrine-metabolic and psychosocial issues, worsened by a long diagnostic delay, requiring a multidisciplinary approach. The scarcity of reference Sleep Centres forces the patient and family to travel for seeking medical consultations, increasing the economic and psychosocial burden of the disease. Growing evidence suggests that Telemedicine may facilitate patient access to sleep consultations and its non-inferiority in terms of patient satisfaction, adherence to treatment, and symptom improvement for sleep disorders. However, Telemedicine clinical and economic benefits for patients with narcolepsy are still unknown. METHODS: TENAR is a two-part project, including: 1. a cross-sectional study (involving 250 children and adults with suspected narcolepsy) evaluating the accuracy of Teletriage (i.e., a synchronous live interactive sleep assessment through a Televisit) for narcolepsy diagnosis compared to the reference standard; and 2. a two-arm, parallel, open randomized controlled trial (RCT) to demonstrate the non-inferiority of the multidisciplinary care of narcolepsy through Televisits versus standard care. In this RCT, 202 adolescents (> 14 y.o.) and adults with narcolepsy will be randomly allocated (1:1 ratio) either to Televisits via videoconference or to standard in-person outpatient follow-up visits (control arm). The primary outcome is sleepiness control (according to the Epworth Sleepiness Scale). Secondary outcomes are other symptoms control, compliance with treatment, metabolic control, quality of life, feasibility, patient and family satisfaction with care, safety, and disease-related costs. At baseline and at 12 months, patients will undergo neurologic, metabolic, and psychosocial assessments and we will measure primary and secondary outcomes. Primary outcomes will be also measured at 6 months (remotely or in person, according to the arm). DISCUSSION: TENAR project will assess, for the first time, the feasibility, accuracy, efficacy and safety of Telemedicine procedures applied to the diagnosis and the multidisciplinary care of children and adults with narcolepsy. The study may be a model for the remote management of other rare disorders, offering care access for patients living in areas lacking medical centres with specific expertise. TRIAL REGISTRATION: Number of the Tele-multidisciplinary care study NCT04316286. Registered 20 March 2020.

Flow cytometry T cell profiling in a recent-onset narcoleptic type 1 child: a case report.

BACKGROUND: Recent studies have disclosed the involvement of T cells in narcolepsy type 1 pathogenesis. We characterized the T cell subsets distribution in a recent-onset child at two different time points, two months from disease onset (T0) and 10 months later (follow-up), respectively. METHODS: Peripheral blood mononuclear cells (PBMCs) and cerebrospinal fluid (CSF) lymphocytes were characterized by flow cytometry at both evaluations. The distribution of T cell subsets was compared between the two time points, and the fold change was calculated as the CSF/PBMC frequencies ratio. RESULTS: The patient showed a 2-fold increase in the frequency of CD4+ EMRA T cells, and an increase of more than one and a half in the frequency of CD4+ CM T cells in CSF at follow-up compared to T0. Moreover, the distribution of CD4+ EM T cells was slightly increased at T0 compared to follow-up. In PBMCs, the CD4+ and CD8+ T cell subsets showed a slight decrease in CM cells at the follow-up. Finally, the CSF/PBMC fold-change ratio showed a 3-fold increase of CD4+ and CD8+ CM T cells at the follow-up, a rise up to 1.5-fold of CD4+ EMRA subsets and a slight decrease in CD4+ EM T cells. CONCLUSIONS: Our data suggest that variations in the frequency of EM vs. CM of CD4+ and CD8+ T cell subsets in the CSF and in the CSF/PBMC fold change may represent a biological marker of disease progression.

Excessive daytime sleepiness in narcolepsy and central nervous system hypersomnias.

PURPOSE: Excessive daytime sleepiness (EDS) is the core complaint of central nervous system (CNS) hypersomnias. In this mini-review, we summarized EDS features in CNS hypersomnias to provide a guide for differential diagnosis purposes. METHODS: A review of recent literature was performed to provide an update in CNS hypersomnias. RESULTS: At clinical evaluation, narcolepsy patients report a good restorative potential of sleep together with the frequent occurrence of dreaming even during short-lasting naps. These features are mirrored by the neurophysiological evidence of REM sleep at sleep onset (SOREMP) during the Multiple Sleep Latency Test (MSLT), a specific marker. Conversely, patients with idiopathic hypersomnia (IH) complain sleep inertia and prolonged nocturnal sleep. Polysomnographic studies show high sleep propensity on the MSLT or high 24-h total sleep time during continuous monitoring. Patients with insufficient sleep syndrome (ISS) can present with variable clinical EDS features in between narcolepsy and IH. ISS diagnosis is based on the clinical evidence of nocturnal sleep curtailment (weekdays versus vacations) associated with the disappearance of EDS complaint after sleep extension. Polysomnographic data are not required, but when the MSLT is performed, ISS patients can present with SOREMP arising from non-REM stage 2 sleep (vs narcolepsy patients entering into SOREM most frequently from wakefulness). Kleine-Levin Syndrome is characterized by recurrent episodes of enormously prolonged sleep time lasting days associated with abnormal cognition and behavior intermixed by asymptomatic periods, a sleep pattern that can be well documented by actigraphy. CONCLUSIONS: Different CNS hypersomnias present with specific features of EDS are useful to guide the clinician to apply and interpret appropriate neurophysiological investigations.

Cerebrospinal fluid biomarkers of neurodegeneration in narcolepsy type 1.

STUDY OBJECTIVES: To measure the levels of five neurodegenerative biomarkers in the cerebrospinal fluid (CSF) of patients with narcolepsy type 1 (NT1) with variable disease duration. METHODS: Following a standardized protocol of CSF collection and storage, we measured CSF total- and phosphorylated-tau, amyloid-beta 1-40 and 1-42, and neurofilament light chain (NfL) proteins in 30 nonneurological controls and 36 subjects with NT1, including 14 patients with recent disease onset (i.e. ≤12 months, short disease duration group). RESULTS: CSF levels of all biomarkers were similar in NT1 subjects and controls. The comparison between NT1 with short and long disease duration only revealed slightly higher levels of CSF amyloid-beta 1-40 in the former group (median 9,549.5, interquartile range [IQR] 7,064.2-11,525.0 vs. 6,870.0, IQR 5,133.7-9,951.2, p = 0.043). CSF storage time did not influence the levels of the tested biomarkers. CONCLUSIONS: The measurement of CSF total-tau, phosphorylated-tau, amyloid-beta 1-40 and 1-42, and NfL proteins is not informative in NT1.

Development and validation of volumetric absorptive microsampling coupled with UHPLC-MS/MS for the analysis of gamma-hydroxybutyric acid in human blood.

A volumetric microsampling (VAMS) device (20 µl) was evaluated and validated for the analysis of γ-hydroxybutyric acid (GHB) in venous blood using a simple ultra-high-pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method. GHB was extracted from VAMS device by acetonitrile, after a re-hydration step in a temperature-controlled ultrasonic bath at 60°C for 10 min. Chromatographic analysis was carried out on a Kinetex C18 column using 0.1% formic acid in water and acetonitrile as binary gradient mobile phase (from 5 to 95% of acetonitrile from 1 to 2.5 min) at a flow rate of 0.3 ml/min. The VAMS method was fully validated according to current guidelines with satisfactory results in terms of linearity, selectivity, precision, absolute recovery, matrix effect and stability. The linearity was determined from 0.5 to 200 µg/ml and the lower limit of quantitation was 0.5 µg/ml. The novel VAMS-UHPLC-MS/MS method was successfully compared with plasma-based method in a GHB-treated patient as a proof of concept.

Biomarkers for REM sleep behavior disorder in idiopathic and narcoleptic patients.

To search for discriminating biomarkers, 30 patients with idiopathic rapid-eye-movements sleep behavior disorder (iRBD) were compared with 17 patients with RBD within narcolepsy type 1. Both groups underwent extensive examinations, including skin biopsy searching for phosphorylated α-synuclein deposits and whole-night video-polysomnography. Skin biopsy was positive for phosphorylated α-synuclein deposits in 86.7% of iRBD patients and in none of narcoleptic patients. The analysis of video-polysomnographic motor events showed differences in their occurrence throughout the night in the two groups. iRBD and RBD due to narcolepsy do have different clinical and pathological findings, confirming a different pathophysiology.

Cardiovascular autonomic dysfunction, altered sleep architecture, and muscle overactivity during nocturnal sleep in pediatric patients with narcolepsy type 1.

STUDY OBJECTIVES: Arterial blood pressure (ABP) decreases during sleep compared with wakefulness and this change is blunted in mouse models of and adult patients with narcolepsy type 1 (NT1). We tested whether: (1) pediatric patients with NT1 have similar cardiovascular autonomic abnormalities during nocturnal sleep; and (2) these abnormalities can be linked to hypocretin-1 cerebrospinal fluid concentration (CSF HCRT-1), sleep architecture, or muscle activity. METHODS: Laboratory polysomnographic studies were performed in 27 consecutive drug-naïve NT1 children or adolescents and in 19 matched controls. Nocturnal sleep architecture and submentalis (SM), tibialis anterior (TA), and hand extensor (HE) electromyographic (EMG) activity were analyzed. Cardiovascular autonomic function was assessed through the analysis of pulse transit time (PTT) and heart period (HP). RESULTS: PTT showed reduced lengthening during total sleep and REM sleep compared with nocturnal wakefulness in NT1 patients than in controls, whereas HP did not. NT1 patients had altered sleep architecture, higher SM EMG during REM sleep, and higher TA and HE EMG during N1-N3 and REM sleep when compared with controls. PTT alterations found in NT1 patients were more severe in subjects with lower CSF HRCT-1, but did not cluster or correlate with sleep architecture alterations or muscle overactivity during sleep. CONCLUSION: Our results suggest that pediatric NT1 patients close to disease onset have impaired capability to modulate ABP as a function of nocturnal wake-sleep transitions, possibly as a direct consequence of hypocretin neuron loss. The relevance of this finding for cardiovascular risk later in life remains to be determined.