Vitamin D deficiency in type 1 narcolepsy: a reappraisal

Accepted Manuscript Title: Vitamin D deficiency in type 1 narcolepsy: a reappraisal Author: Yves Dauvilliers, Elisa Evangelista, Regis Lopez, Lucie Barateau, Sabine Scholz, Barbara Crastes de Paulet, Bertrand Carlander, Isabelle Jaussent PII: DOI: Reference:

S1389-9457(16)30087-9 http://dx.doi.org/doi: 10.1016/j.sleep.2016.05.008 SLEEP 3097

To appear in:

Sleep Medicine

Received date: Revised date: Accepted date:

8-1-2016 31-3-2016 15-5-2016

Please cite this article as: Yves Dauvilliers, Elisa Evangelista, Regis Lopez, Lucie Barateau, Sabine Scholz, Barbara Crastes de Paulet, Bertrand Carlander, Isabelle Jaussent, Vitamin D deficiency in type 1 narcolepsy: a reappraisal, Sleep Medicine (2016), http://dx.doi.org/doi: 10.1016/j.sleep.2016.05.008. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Vitamin D deficiency in type 1 narcolepsy: a reappraisal Yves Dauvilliers a,b,*, Elisa Evangelista a, Regis Lopez a,b, Lucie Barateau a,b, Sabine Scholz a, Barbara Crastes de Paulet c, Bertrand Carlander a, Isabelle Jaussent b

a

National Reference Network for Narcolepsy, Department of Neurology, Hôpital Gui-de-Chauliac, CHU

Montpellier, France b

Inserm U1061, University of Montpellier 1, Montpellier, France

c

Laboratoire de biochimie-hormonologie, Hôpital Lapeyronie, CHU Montpellier, France

*Corresponding author. Service de Neurologie, Hôpital Gui-de-Chauliac, 80 avenue Augustin Fliche, 34295 Montpellier cedex 5, France. Tel.: (33) 4 67 33 74 78; fax: (33) 4 67 33 72 85. E-mail address: [email protected] (Y. Dauvilliers)

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Highlights

-

Narcolepsy type 1 (NT1) is considered to be an atypical immune-mediated disease in which environmental factors might play a major role.

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Vitamin D levels and the fraction of subjects with vitamin D deficiency (serum level <75 or 50 nmol/L) did not differ between patients with narcolepsy and controls.

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No association was found between vitamin D deficiency, narcolepsy duration and severity, treatment and Pandemrix® vaccination.

Abstract Objective: Narcolepsy type 1 (NT1) is considered to be an immune-mediated disease in which environmental factors, such as vitamin D, might play a major role. The association between NT1 and vitamin D deficiency has previously been reported. The aim of this case-control study was to reassess vitamin D levels in a large clinic-based adult and paediatric population of patients with NT1 by considering several potential confounding factors. Methods: The serum level of 25-hydroxyvitamin D (25OHD) was measured in 174 Caucasian patients with NT1 and 174 controls. Demographic and clinical features, body mass index (BMI), Pandemrix® vaccination, age and season at the time of blood sampling were recorded. Between-group comparisons were made using univariate and multivariate logistic regression analyses. When appropriate, interaction terms were tested using the Wald Chi-squared test. Results: Age, BMI and season of blood sampling were different between groups. Conversely, the 25OHD level and fraction of subjects with vitamin D deficiency (serum level <75 nmol/L: 46.6% of patients vs 48.3% of controls; <50 nmol/L: 20.7% vs 17.2%) did not differ between patients with NT1 and controls. Overall, vitamin D deficiency was more frequent in men, obese subjects and in samples collected in winter, without any association with NT1. In the patients’ group, no significant association was found between vitamin D deficiency, NT1 duration and severity, treatment and Pandemrix® vaccination. Conclusions: Vitamin D levels were not associated with NT1 in a large case-control population when potential demographic and clinical confounding factors were taken into account.

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Keywords:

Comment [JM1]: There is a maximum of 6 keywords allowed, please delete one as you have 7

Narcolepsy Vitamin D Hypocretin/orexin Sleepiness Autoimmunity Cataplexy Vaccination

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Introduction Narcolepsy with cataplexy, which was recently renamed narcolepsy type 1 (NT1), is a sleep disorder with symptom onset mostly in childhood and adolescence [1,2]. Narcolepsy type 1 is caused by the loss of hypocretin (HCRT)-producing neurons in the hypothalamus [1]. Although the precise aetiology remains unknown, NT1 is strongly associated with HLA-DQB1*06:02 [3] and also other HLA-DPB1 and HLA Class I alleles [4], polymorphisms in the T-cell receptor α locus [5] and the purinergic receptor subtype 2Y11 [6]. Environmental factors also play a significant role, and associations have been reported with streptococcal infections [7], the 2009-2010 H1N1 influenza pandemic [8] and vaccination with the AS03 adjuvant H1N1 pandemic Pandemrix® vaccine [9-12]. These findings suggest that NT1 could be an immune-mediated disorder leading to the selective loss of HCRT-containing neurons [13,14]. However, the evidence that these triggers are causative agents for NT1 remains indirect and relatively weak [13,14]. The precise immune-related mechanisms in NT1 remain poorly understood and little is known about the key environmental factors.

Vitamin D is a steroid hormone, and its active metabolite 1,25-(OH)2D is the ligand for a transcription factor and intracellular receptor that is called ‘vitamin D receptor’ and is expressed by brain (microglia) and circulating immune cells [15,16]. It has been suggested that vitamin D is a major environmental factor implicated in the aetiology of many autoimmune diseases, including those restricted to the central nervous system (CNS) [17]. Vitamin D regulates the expression of major histocompatibility complex (MHC) class II genes, modulates the activity of regulatory T lymphocyte cells and modifies the balance between Th2 and Th1 lymphocytes [18-20]. Robust associations between sunlight and vitamin D in multiple sclerosis and type 1 diabetes suggest that vitamin D has a key role in the development of autoimmune diseases [16,21,22] that share potential T-cell-mediated mechanisms with NT1 [14,23]. In multiple sclerosis, lower vitamin D levels increase the disease risk, the relapse rate, and the specific disease activity and disability measures [22,24,25]. Moreover, in a small-size, case-control study, it was found that vitamin D deficiency was more frequent in adult patients with NT1 than in healthy controls in crude and adjusted models [26]. However, there was no association with NT1 duration and severity or treatment intake.

The aim of the present study was to confirm and extend these results in a larger clinic-based case-control population of adults and children with a definite diagnosis of NT1. Using the same method of measurement as before, the serum level of 25-hydroxyvitamin D (25OHD) was analysed in patients with NT1 and control subjects by taking into account the main factors that might influence vitamin D levels, NT1 clinical features and the impact of vaccination with the Pandemrix® vaccine.

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Methods Subjects This study included 174 European Caucasian patients with NT1 (108 males and 66 females, median age 32.5 years, range 6–68 years). Diagnosis of NT1 was based on the following criteria: history of clear-cut cataplexy and mean multiple sleep latency test (MSLT) ≤8 minutes with ≥2 sleep onset REM periods (SOREMPs) [27,28] or cerebrospinal fluid (CSF) HCRT-1 deficiency (<110 pg/mL), according to the revised International Classification of Sleep Disorders (ICSD-3) [29]. Sixty-seven patients had a lumbar puncture, including the patients with atypical MSLT results (n=7) or without typical cataplexy (n=3), and all had CSF HCRT-1 levels <110 pg/mL. None of the patients presented with a psychiatric disorder, based on the DSM-V criteria, or any other significant comorbid conditions. Age, sex, body mass index (BMI) (normal: <25 kg/m2; overweight: ≥25 kg/m2; obese: ≥30 kg/m2) [30], season of blood sampling, NT1 clinical data (age at onset, disease duration, Epworth Sleepiness Scale (ESS) score for adults or Adapted Epworth Sleepiness Scale (AESS) score for children, cataplexy frequency scale [2], hypnagogic hallucinations and sleep paralysis), polysomnographic data (mean sleep latency and number of SOREMPs in the MSLT, and apnoea/hypopnea index, AHI) and treatment at the time of the study were recorded for all patients. Fifteen patients (8.62%) were vaccinated with the Pandemrix® vaccine before narcolepsy onset.

Controls (174 European Caucasian subjects, 119 males and 55 females, median age 36.0 years, range 5-87 years) were recruited from the general population in the same area in the South of France via advertisement, during the same period. Controls were community-dwelling subjects without any significant medical, neurological or psychiatric disease. Demographic data (age and sex), BMI, season of blood sampling, ESS/AESS scores were collected. Among the controls, 17.3% had an ESS >10, but further investigations (polysomnography, even prolonged for 24 hours, and MSLT) ruled out the diagnosis of central hypersomnia. None of the subjects included in the previous study on NT1 and vitamin D [24] were enrolled in the present study.

All subjects and the parents of children aged <18 years signed a written informed consent to participate in the study, which was approved by the local ethics committee. According to French law, the biological sample collection was registered at the ‘Ministère de l’Enseignement Superieur et de la Recherche’ (Number DC-2008- 417).

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Blood analysis Venous blood was sampled between 07:00 and 08:00 after overnight fasting, according to standardised procedures. Serum samples from patients and controls were handled similarly and frozen immediately for further analysis. The serum 25OHD level, which is representative of the overall vitamin D stored in the body (25-hydroxyvitamin D ergocalciferol (D2) plus cholecalciferol (D3)) [15], was measured by radioimmunoassay (25OH Vitamin D RIA, IDS, Immunodiagnostic Systems, Boldon, UK). The inter-assay coefficient of variation was <8.2% and the intra-assay variation coefficient was <6.1%. Samples were run in separate, single assays. Two commonly accepted international clinical thresholds for vitamin D deficiency and insufficiency, respectively, (serum 25OHD level <50 and <75 nmol/L) were used [22,31].

Statistical analysis Percentages were used for categorical variables and medians and ranges for quantitative variables; the distribution of continuous variables was mostly skewed, according to the Shapiro-Wilk test. Univariate comparisons between controls and patients with NT1 were made using logistic regression analysis and quantified with odds ratios (OR) and their 95% confidence intervals (CI). The serum 25OHD levels (continuous variable) were divided into quartiles and tertiles, and were also based on the clinical thresholds of 50 and 75 nmol/L. Multivariate logistic regression analyses were performed on clinical and demographic variables with p<0.1 in univariate analysis. When appropriate, interaction terms were tested using the Wald Chi-squared test given by the logistic regression model. Significance level was set at p<0.05. Statistical analyses were performed using the SAS software, version 9.4 (SAS Institute, Cary, NC, USA).

Results The median age at disease onset in the NT1 group was 18 years (range 4–61 years) and disease duration was 10.5 years (range 0–73 years). Among the patients with NT1, 65.9% reported hypnagogic hallucinations, 55.1% sleep paralysis, 34.3% REM sleep behaviour disorder, and 8.9% severe obstructive sleep apnoea syndrome (AHI >30/hours). At the time of blood sampling, 34% of patients with NT1 were treated with psychostimulants or anticataplectic drugs.

Compared with the controls, patients with NT1 were significantly older and more overweight/obese (BMI >30kg/m2: 15.9% vs 9.3% of controls) (Table 1). As expected, patients with NT1 had higher ESS/AESS

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scores (95.6% of NT1 patients had a score >10 compared with 17.3% of controls). Blood sampling in winter was significantly more frequent in the control group than in the patient group.

The serum level of 25OHD was not significantly different between patients with NT1 and controls in crude association or after adjustment for age, BMI and season of blood sampling (Table 2, Fig. 1). No significant between-group difference was observed when vitamin D deficiency was defined according to the two thresholds (<75 nmol/L: 46.6% of patients vs 48.3% of controls; <50 nmol/L: 20.7% of patients vs 17.2% of controls), or when serum 25OHD levels were divided into quartiles and tertiles of the whole sample (Table 2). A very low vitamin D concentration (<25 nmol/L) was found in five patients with NT1 and two controls.

A comparison of all subjects (patients and controls) with low (<75, n=165) and normal vitamin D levels (≥75, n=183) indicated that vitamin D deficiency was more frequent in men and obese individuals, and in blood samples taken in winter (Table 3). No significant association was found between vitamin D level and age, disease status or ESS score. These findings did not change significantly, except for the disappearance of the sex difference, when vitamin D deficiency was defined using the cut-off of 50 nmol/L instead of 75 nmol/L (data not shown).

Further analyses, using only the data of patients with NT1 to unravel potential intrinsic risk factors for vitamin D deficiency, did not highlight any significant association, except for the season of blood sampling (p=0.008), between vitamin D levels (cut off of 75 nmol/L) and demographic features (age, sex and BMI), clinical data (age at onset, disease duration, ESS/AESS, frequency of cataplexy, hypnagogic hallucinations, sleep paralysis, REM sleep behaviour disorder and treatment), neurophysiological features (MSLT latency, number of SOREMPs, AHI) and H1N1 influenza vaccine (Table 4). When the cut-off of 50 nmol/L was used, the season of blood sampling (p=0.005) and BMI was associated with the vitamin D status (p=0.04).

Finally, to try to understand why the current study did not confirm the previous finding of an association between vitamin D deficiency and NT1 [26], despite having used the same method of biological measurement, the populations (patients and controls) of the two studies were compared. Between-patient group differences were found. Patients with NT1 in the current study were more likely to be men, sleepier (ESS >10: 95.6% vs 87.2%, p=0.05), with more frequent cataplexy (median frequency at 5.00 (0.00–5.00) vs 3.00 (1.00–5.00), p=0.0003). Moreover, the median vitamin D level was higher in the patients of the

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current study (77.21 (3.73–437.25) vs 59.80 (24.05–124.03), p=0.003). Indeed, vitamin D level was below the cut-off of 75 nmol/L only in 46.6% of the current patients compared with 70% in the previous study (p=0.004). Differences between control groups were also found. Control subjects in the present study were more likely to be men, and blood sampling was performed more often in winter and autumn (p=0.0001). However, vitamin D levels were not different between control groups, using either the median values (77.46 (21.10–209.90) vs 80.80 (26.88–167.48), or the threshold of <75 nmol/L (48.3 vs 48.15%).

Discussion In this study the vitamin D levels in serum samples were assessed from 174 patients with NT1 and 174 healthy controls by taking into account potential demographic and clinical confounding factors; no significant between-group differences were found.

It has previously been reported in a smaller population sample that there are a significantly lower vitamin D levels in adult patients with NT1 (n=51) compared with age-matched and sex-matched controls (n=55) [26]. The association between vitamin D level and NT1 persisted after adjustment for potential confounding factors such as BMI and season of blood sampling. However, there was no association found between vitamin D levels, age at disease onset, disease duration, ESS and MSLT results, cataplectic attack frequency and NT1 treatment.

The present study did not find any significant association between NT1 and vitamin D deficiency using either validated cut-off values (<75 or <50 nmol/L) or by dividing the serum 25OHD levels into quartiles or tertiles. The well-known association between vitamin D deficiency, BMI (obesity) and season of blood sampling (winter time) was confirmed [15,16]. However, and as in the previous study, there was no association found between vitamin D levels and disease features. More precisely, no association was found between vitamin D, age, age at disease onset, disease duration at the time of blood sampling, and treatment. Moreover, no association was found between vitamin D levels, NT1 and exposure to the Pandemrix® vaccine.

Altogether, the discrepancy of these two studies may be due to some demographic and clinical differences between populations. In the first study, the sample was smaller, the dispersion of vitamin D level higher,

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with a large 95% confidence interval that tends to give less precise estimates of the effects. In the current study, patients had a higher median vitamin D level and more severe NT1 symptomatology.

Moreover, in line with the current results, and to the best of the authors’ knowledge, no epidemiological study has reported geographical variations in NT1 prevalence in function of sunlight exposure. Conversely, several recent studies have confirmed that vitamin D deficiency is common and its prevalence is increasing in the general population. In the last decade, 41.6% of adult Americans had vitamin D insufficiency [32] and the lowest range of 25OHD level has decreased from 75 to 50 nmol/L in recent years [32,33]. Accordingly, vitamin D deficiency or insufficiency (ie <50 or 75 nmol/L) is frequently reported in normal controls, depending mainly on sunshine exposure and less on food intake, with a frequent overlap between normal and pathological conditions.

The lack of association between vitamin D levels and NT1 should give some clues regarding the pathogenesis of NT1. Briefly, immunity is based on two distinct pathways: the innate and the adaptive immune systems. Cells of the innate (macrophage and dendritic cells) and the adaptive (CD4+ T, CD8+ T, B and natural killer lymphocytes) immune systems have the ability to convert 25(OH)D into 1,25(OH)2D and express the vitamin D receptor [34]. The vitamin D pathway participates in regulating the innate immune system via the stimulation of the Toll-like receptor 2 and the reinforcement of the macrophage defence mechanisms [35]. In contrast, the action of vitamin D is generally suppressive on the adaptive immune system with the decrease of T-cell proliferation and Th1 differentiation by direct activation on T-cell receptors, the decrease in cytokine secretion (interferon and interleukin-2) by CD4+ T-cells, and the cytotoxicity of CD8+ T-cells [36]. Of interest, a recent study highlighted the role of pro-inflammatory cytokines, and especially interferon-γ, in narcolepsy being potential key players in the immune mechanism that triggers NT1 [37]. In contrast to NT1, robust associations were found between vitamin D and multiple sclerosis [20-22] that may be driven by the Th-17 cytokine pathway. Indeed, vitamin D has been found to suppress Th-17-driven cytokine responses [38], without any data suggesting CNS specific IL-17 network involvement in NT1 [37]. Given the variable knowledge of the relationships of vitamin D to each of the autoimmune diseases, it remains impossible at this stage to individualise the key immune pathway that explains the lack of association with vitamin D levels in NT1. However, recent studies have strongly supported that the supposed autoimmune basis in NT1 differs from most of the known autoimmune diseases, in terms of strong HLA DQB1*06:02 association and T-cell receptor alpha polymorphisms, with potential involvement of cytotoxic CD8+ T cells [13,14,23,39].

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The present cross-sectional study had some limitations and strengths. First, the patients with NT1 were not HLA-matched to normal controls. However, as the HLA DQB1*0602 allele is present in 20% of Caucasian controls, an HLA-adjusted comparison would have required 870 control subjects, and this was beyond the study resources. Second, some controls were sleepy with an ESS >10; however, none of them had a hypersomnolence disorder and no association was found between vitamin D level and ESS score. Third, the controls were not age-matched and BMI-matched with patients with NT1, but adjustment for these factors did not modify the results. Fourth, serum samples were taken after variable disease durations and this could have decreased the opportunity to detect vitamin D deficiency at disease onset. However, no association was found between 25OHD level, age, age at disease onset and disease duration.

The study had several strengths. It had a large case-control population, with children and adults, all of Caucasian ethnicity and all residing in southern France (a sunny area), without any overlap with the subjects included in the previous narcolepsy vitamin D study [26]. All were recruited at a single reference sleep centre for narcolepsy to avoid potential uncertainties in disease phenotyping, blood sampling and handling. Sampling conditions (fasting, time of the day and handling rapidity) were similar for all subjects. All 25OHD level measurements were performed in the same laboratory using the same validated method. Finally, several potential confounding factors were taken into account, particularly demographic, clinical and neurophysiological features, treatment, season of blood sampling and exposure to the Pandemrix® vaccine.

In conclusion, this large case-control population study demonstrated that vitamin D level is not associated with NT1, after taking into account potential demographic and clinical confounding factors. This finding suggests that vitamin D level is not an informative biomarker in patients with NT1 and that vitamin D supplementation in NT1 might not be required.

Conflict of interest: Y Dauvilliers has been an invited speaker and consultant for UCB pharma, JAZZ, Bioprojet and Mundipharma. Other co-authors had no conflict of interest.

Acknowledgement: Support for this research was provided by the French Ministry of Research and Higher Education, Project Agence Nationale de la Recherche-2014-ImmunitySleep, and Aviesan-ITMO 2014 BioNarcoImmunity

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References 1

Dauvilliers Y, Arnulf I, Mignot E. Narcolepsy with cataplexy. Lancet. 2007;369:499–511.

2

Dauvilliers Y, Montplaisir J, Molinari N, et al. Age at onset of narcolepsy in two large populations of patients in France and Quebec. Neurology. 2001;57:2029–33.

3

Mignot E. Complex HLA-DR and -DQ interactions confer risk of narcolepsy-cataplexy in three ethnic groups. Am J Hum Genet. 2001;68:686–99.

4

Ollila HM, Ravel J-M, Han F, et al. HLA-DPB1 and HLA class I confer risk of and protection from narcolepsy. Am J Hum Genet. 2015;96:136–46.

5

Hallmayer J. Narcolepsy is strongly associated with the T-cell receptor alpha locus. Nat Genet. 2009;41:708–11.

6

Kornum BR, Kawashima M, Faraco J, et al. Common variants in P2RY11 are associated with narcolepsy. Nat Genet. 2011;43:66–71.

7

Aran A, Lin L, Nevsimalova S, et al. Elevated anti-streptococcal antibodies in patients with recent narcolepsy onset. Sleep. 2009;32:979–83.

8

Han F, Lin L, Warby SC, et al. Narcolepsy onset is seasonal and increased following the 2009 H1N1 pandemic in China. Ann Neurol. 2011 Sep;70:410–7.

9

Nohynek H, Jokinen J, Partinen M, et al. AS03 adjuvanted AH1N1 vaccine associated with an abrupt increase in the incidence of childhood narcolepsy in Finland. PloS One. 2012;7:e33536.

10

Wijnans L, Lecomte C, de Vries C, et al. The incidence of narcolepsy in Europe: before, during, and after the influenza A(H1N1)pdm09 pandemic and vaccination campaigns. Vaccine. 2013;31:1246–54.

11

Partinen M, Saarenpää-Heikkilä O, Ilveskoski I, et al. Increased incidence and clinical picture of childhood narcolepsy following the 2009 H1N1 pandemic vaccination campaign in Finland. PloS One. 2012;7:e33723.

12

Dauvilliers Y, Arnulf I, Lecendreux M, et al. Increased risk of narcolepsy in children and adults after pandemic H1N1 vaccination in France. Brain . 2013;136:2486–96.

13

Partinen M, Kornum BR, Plazzi G, et al. Narcolepsy as an autoimmune disease: the role of H1N1 infection and vaccination. Lancet Neurol. 2014;13:600–13.

14

Liblau RS, Vassalli A, Seifinejad A, et al. Hypocretin (orexin) biology and the pathophysiology of narcolepsy with cataplexy. Lancet Neurol. 2015;14:318–28.

15

Adams JS, Hewison M. Update in vitamin D. J Clin Endocrinol Metab. 2010;95:471–8.

16

Rosen CJ, Adams JS, Bikle DD, et al. The nonskeletal effects of vitamin D: an Endocrine Society scientific statement. Endocr Rev. 2012;33:456–92.

17

Eyles DW, Smith S, Kinobe R, et al. Distribution of the vitamin D receptor and 1 alpha-hydroxylase in human brain. J Chem Neuroanat. 2005;29:21–30.

18

Edfeldt K, Liu PT, Chun R, et al. T-cell cytokines differentially control human monocyte antimicrobial responses by regulating vitamin D metabolism. Proc Natl Acad Sci U S A. 2010;107:22593–8.

Page 11 of 22

19

Von Essen MR, Kongsbak M, Schjerling P, et al. Vitamin D controls T cell antigen receptor signaling and activation of human T cells. Nat Immunol. 2010;11:344–9.

20

Ramagopalan SV, Maugeri NJ, Handunnetthi L, et al. Expression of the multiple sclerosis-associated MHC class II Allele HLA-DRB1*1501 is regulated by vitamin D. PLoS Genet. 2009;5:e1000369.

21

Ebers GC. Environmental factors and multiple sclerosis. Lancet Neurol. 2008;7:268–77.

22

Pierrot-Deseilligny C, Souberbielle J-C. Is hypovitaminosis D one of the environmental risk factors for multiple sclerosis? Brain J Neurol. 2010;133:1869–88.

23

Degn M, Kornum BR. Type 1 narcolepsy: a CD8(+) T cell-mediated disease? Ann N Y Acad Sci. 2015;1351:80–8.

24

Mowry EM, Krupp LB, Milazzo M, et al. Vitamin D status is associated with relapse rate in pediatriconset multiple sclerosis. Ann Neurol. 2010;67:618–24

25. Mowry EM, Waubant E, McCulloch CE, et al. Vitamin D status predicts new brain magnetic resonance imaging activity in multiple sclerosis. Ann Neurol. 2012;72:234–40. 26. Carlander B, Puech-Cathala AM, Jaussent I, et al. Low vitamin D in narcolepsy with cataplexy. PloS One. 2011;6:e20433. 27

Iber C, Ancoli-Israel S, Chesson A, et al., for the American Academy of Sleep Medicine. The AASM manual for the scoring of sleep and associated events; rules, terminology and technical specifications. 1st ed. Westchester, IL: American Academy of Sleep Medicine; 2007.

28

Littner MR, Kushida C, Wise M, et al. Practice parameters for clinical use of the multiple sleep latency test and the maintenance of wakefulness test. Sleep. 2005;28:113–21.

29

AASM: American Academy of Sleep Medicine. ICSD-3: International Classification of Sleep Disorders, 3rd ed. American Academy of Sleep Medicine. 2014.

30

National Heart, Lung and Blood Institute. Clinical Guidelines on the Identification, Evaluation, and Treatment of Overweight and Obesity in Adults. 1998

31

Souberbielle J-C, Body J-J, Lappe JM, et al. Vitamin D and musculoskeletal health, cardiovascular disease, autoimmunity and cancer: Recommendations for clinical practice. Autoimmun Rev. 2010;9:709–15.

32

Looker AC, Pfeiffer CM, Lacher DA, et al. Serum 25-hydroxyvitamin D status of the US population: 1988-1994 compared with 2000-2004. Am J Clin Nutr. 2008;88:1519–27.

33

Yetley EA. Assessing the vitamin D status of the US population. Am J Clin Nutr. 2008;88:558S – 564S.

34. Mora JR, Iwata M, von Andrian UH. Vitamin effects on the immune system: vitamins A and D take centre stage. Nat Rev Immunol. 2008 ;8:685-98.

35. Liu PT, Stenger S, Li H, et al. Toll-like receptor triggering of a vitamin D-mediated human antimicrobial response. Science. 2006 ;311:1770-3 36. Peelen E, Knippenberg S, Muris AH, et al. Effects of vitamin D on the peripheral adaptive immune system: a review. Autoimmun Rev. 2011 ;10:733-43.

Page 12 of 22

37. Lecendreux M, Libri V, Jaussent I, et al. Impact of cytokine in type 1 narcolepsy: Role of pandemic H1N1 vaccination ? J Autoimmun. 2015;60:20-31 38. Ouyang W, Kolls JK, Zheng Y. The biological functions of T helper 17 cell effector cytokines in inflammation. Immunity. 2008;28:454-67

39. Dauvilliers Y, Bauer J, Rigau V,et al. Hypothalamic immunopathology in anti-Ma-associated diencephalitis with narcolepsy-cataplexy. JAMA Neurol. 2013;70:1305-10.

Fig. 1. Distribution of the serum level of 25OHD in patients with NT1 (dark grey) and controls (light grey) (n=174/group) without any significant between-group difference.

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Table 1. Demographic and clinical variables of controls and patients with narcolepsy type 1 (NT1).

Variable

Controls

Patients with NT1

N=174

N=174

n

%

n

%

OR [95% CI]

p

Sex Male

108

62.07

119

68.39

1

Female

66

37.93

55

31.61

0.76 [0.49-1.18]

Age (years)*

0.22

32.50 [6-68]

36 [5-87]

1.01 [1.00-1.02] 0.04

Body Mass Index (kg/m )*

22.80 [14.20-62.00]

25.50 [15.00-45.00]

1.04 [1.00-1.08] 0.04

Epworth sleepiness scale*

6.00 [0.00-22.00]

18.00 [5.00-24.00]

1.55 [1.42-1.70] <0.0001

Winter

64

36.78

42

24.14

1

Spring

26

14.94

44

25.29

2.58 [1.38-4.80]

Summer

28

16.09

42

24.14

2.29 [1.23-4.23]

Autumn

56

32.18

46

26.44

1.25 [0.72-2.17]

2

Season of blood sampling 0.005

* Median values [range]

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Table 2. Serum 25-hydroxyvitamin D (25OHD) levels in controls and patients with narcolepsy type 1 (NT1).

Controls

Patients with NT1

N=174 Variable

n

N=174

%

n

%

OR [95% CI]

p

OR [95% CI] (1) p (1)

77.46 [21.10-209.90]

77.21 [3.73-437.25]

1.06 [0.84-1.33] 0.65

0.98 [0.761.27]

0.86

<75

84

48.28

81

46.55

1

1

0.40

≥75

90

51.72

93

53.45

1.07 [0.70-1.63]

<50

30

17.24

36

20.69

1

≥50

144

82.76

138

79.31

0.80 [0.47-1.37]

<53.175

39

22.41

47

27.01

1

[53.175-77.338]

48

27.59

40

22.99

0.69 [0.38-1.26]

Median 25OHD (nmol/L) (2) 25(OH)D (nmol/L)

0.75

0.81 [0.501.32]

25(OH)D (nmol/L) 0.41

1

0.15

0.64 [0.351.17]

25(OH)D (nmol/L) (quartile) 0.64

1

0.33

0.61 [0.321.18]

Page 15 of 22

Controls

Patients with NT1

N=174 Variable

n

N=174

%

n

%

OR [95% CI]

p

OR [95% CI] (1) p (1)

[77.338-101.888]

45

25.86

42

24.14

0.77 [0.43-1.41]

0.59 [0.311.14]

≥101.888

42

24.14

45

25.86

0.89 [0.49-1.62]

0.58 [0.291.15]

<61.125

55

31.61

60

34.48

1

[61.125-92.50]

62

35.63

54

31.03

0.80 [0.48-1.34]

0.69 [0.391.22]

≥92.50

57

32.76

60

34.48

0.96 [0.58-1.62]

0.68 [0.371.24]

25(OH)D (nmol/L) (tertile)

(1)

Adjustment for age, BMI, and season of blood sampling

(2)

OR for 50 nmol/L increased

0.66

1

0.36

Page 16 of 22

Table 3. Demographic and clinical variables and season of blood sampling in the whole population divided based on the serum 25-hydroxyvitamin D (25OHD) level (75 nmol/L threshold)

25OHD (nmol/L)

Variable

<75

≥75

N=165

N=183

n

%

n

%

OR [IC à 95%]

p

Controls

84

50.91

90

49.18

1

0.75

NT1

81

49.09

93

50.82

1.07 [0.70; 1.63]

Male

121

73.33

106

57.92

1

Female

44

26.67

77

42.08

2.00 [1.27; 3.14]

Disease status

Sex

Age (years)*

0.003

30.00 [6.00-87.00]

37.00 [5.00-78.00] 1.01 [1.00; 1.02]

0.12

<18

33

20.00

24

13.11

1

0.09

≥18

132

80.00

159

86.89

1.66 [0.93; 2.94]

Age (years)

BMI (kg/m2)*

24.30 [14.2062.00]

23.35 [15.0038.10]

0.93 [0.89; 0.97]

0.0007

<25

79

51.63

108

60.67

1

0.001

25-29

43

28.10

59

33.15

1.00 [0.62; 1.64]

≥30

31

20.26

11

6.18

0.26 [0.12; 0.55]

BMI (kg/m2)

Page 17 of 22

25OHD (nmol/L) <75

≥75

N=165

N=183

Variable

n

%

n

%

OR [IC à 95%]

p

Epworth sleepiness scale*

14.00 [0.00-24.00]

14.00 [1.00-24.00] 1.01 [0.97; 1.04]

0.73

<11

52

40.63

70

41.42

1

0.89

≥11

76

59.38

99

58.58

0.97 [0.61; 1.54]

Winter

73

44.24

33

18.03

1

Spring

32

19.39

38

20.77

2.63 [1.41; 4.91]

Summer

19

11.52

51

27.87

5.94 [3.04; 11.6]

Autumn

41

24.85

61

33.33

3.29 [1.86; 5.82]

Epworth sleepiness scale

Season of blood sampling <0.0001

* Median values [range]

Page 18 of 22

Table 4. Demographic and clinical variables and season of blood sampling in patients with NT1 divided based on the serum 25-hydroxyvitamin D (25OHD) level (75 nmol/L threshold)

25OHD (nmol/L)

Variable

<75

≥75

N=81

N=93

n

%

n

%

OR [IC à 95%]

p

Male

59

72.84

60

64.52

1

0.24

Female

22

27.16

33

35.48

1.47 [0.77; 2.82]

Sex

Age (years)*

36.00 [6.0087.00]

36.00 [5.0078.00]

1.00 [0.98; 1.01]

0.48

<18

19

23.46

18

19.35

1

0.51

≥18

62

76.54

75

80.65

1.28 [0.62; 2.64]

Age (years)

BMI (kg/m2)*

26.00 [17.0045.00]

25.00 [15.0038.10]

0.94 [0.88; 1.00]

0.05

<25

31

38.75

43

47.78

1

0.18

25-29

32

40.00

37

41.11

0.83 [0.43; 1.61]

≥30

17

21.25

10

11.11

0.42 [0.17; 1.05]

BMI (kg/m2)

Epworth sleepiness scale*

18.00 [5.0024.00]

19.00 [6.0024.00]

1.02 [0.94; 1.10]

0.70

<11

2

2.94

5

5.56

1

0.44

≥11

66

97.06

85

94.44

0.52 [0.10; 2.74]

Epworth sleepiness scale

Page 19 of 22

25OHD (nmol/L)

Variable

<75

≥75

N=81

N=93

n

%

n

%

OR [IC à 95%]

p

Winter

28

34.57

14

15.05

1

0.008

Spring

21

25.93

23

24.73

2.19 [0.92;5.24]

Summer

12

14.81

30

32.26

5.00 [1.98; 12.6]

Autumn

20

24.69

26

27.96

2.60 [1.09; 6.19]

No

77

95.06

82

88.17

1

Yes

4

4.94

11

11.83

2.58 [0.79; 8.45]

Season of blood sampling

Pandemrix® exposure 0.12

Age at disease onset*

17.00 [5.0051.00]

18.00 [4.0061.00]

1.00 [0.97; 1.03]

0.97

Disease duration*

10 [0-73]

11 [0-63]

1.00 [0.98; 1.01]

0.65

Cataplexy frequency*

5.00 [1.00-5.00]

4.00 [0.00-5.00]

0.89 [0.69; 1.14]

0.35

No

34

61.82

33

70.21

1

0.37

Yes

21

38.18

14

29.79

0.69 [0.30; 1.57]

No

24

32.43

30

35.71

1

Yes

50

67.57

54

64.29

0.86 [0.45; 1.67]

Clinical REM behaviour disorder

Hypnagogic hallucination 0.66

Page 20 of 22

25OHD (nmol/L)

Variable

<75

≥75

N=81

N=93

n

%

n

%

OR [IC à 95%]

p

No

32

43.24

39

46.43

1

0.69

Yes

42

56.76

45

53.57

0.88 [0.47; 1.65]

Multiple sleep latency test, latency (minutes)*

4.50 [0.55-14.00] 3.60 [0.6014.50]

0.95 [0.85; 1.05]

0.30

Number of SOREMPs*

4.00 [0.00-5.00]

4.00 [0.00-5.00]

0.83 [0.61; 1.12]

0.22

<5

38

54.29

54

62.07

1

0.10

[5-15]

11

15.71

21

24.14

1.34 [0.58; 3.11]

[15-30]

12

17.14

7

8.05

0.41 [0.15; 1.14]

≥30

9

12.86

5

5.75

0.39 [0.12; 1.26]

No

50

61.73

64

69.57

1

Yes

31

38.27

28

30.43

0.71 [0.38; 1.33]

Sleep paralysis

Apnoea hypopnea index (per hour)

Treatment at the time of the study 0.28

** Median values [range]

Page 21 of 22

Page 22 of 22