The dementia-associated APOE ε4 allele is not associated with REM sleep behavior disorder

Accepted Manuscript

The dementia-associated APOE ε4 allele is not associated with REM sleep behavior disorder

Ziv Gan-Or, MD, PhD, Jacques Y. Montplaisir, MD, PhD, Jay P. Ross, BSc, Judes Poirier, PhD, Simon C. Warby, PhD, Isabelle Arnulf, MD, PhD, Stephanie Strong, BSc, Yves Dauvilliers, MD, PhD, Claire S. Leblond, PhD, Michele T.M. Hu, MBBS, FRCP, PhD, Birgit Högl, MD, Ambra Stefani, MD, Christelle Charley Monaca, MD, PhD, Valérie Cochen De Cock, MD, PhD, Michel Boivin, PhD, Luigi Ferini-Strambi, MD, PhD, Giuseppe Plazzi, MD, PhD, Elena Antelmi, MD, Peter Young, MD, Anna Heidbreder, MD, Thomas R. Barber, MA, MBBS, MRCP, Samuel G. Evetts, BSc Hons, MSc, Michal Rolinski, BM BCh, BA Hons, MRCP, Patrick A. Dion, PhD, Alex Desautels, MD, PhD, Jean-François Gagnon, PhD, Nicolas Dupré, MD, MSc, Ronald B. Postuma, MD, MSc, Guy A. Rouleau, MD, PhD

PII: S0197-4580(16)30242-1

DOI: 10.1016/j.neurobiolaging.2016.10.002 Reference: NBA 9741

To appear in: Neurobiology of Aging Received Date: 18 August 2016 Revised Date: 22 August 2016 Accepted Date: 1 October 2016

Please cite this article as: Gan-Or, Z., Montplaisir, J.Y., Ross, J.P., Poirier, J., Warby, S.C., Arnulf, I., Strong, S., Dauvilliers, Y., Leblond, C.S., Hu, M.T.M., Högl, B., Stefani, A., Monaca, C.C., De Cock, V.C., Boivin, M., Ferini-Strambi, L., Plazzi, G., Antelmi, E., Young, P., Heidbreder, A., Barber, T.R, Evetts, S.G., Rolinski, M., Dion, P.A., Desautels, A., Gagnon, J.-F., Dupré, N., Postuma, R.B., Rouleau, G.A., The dementia-associated APOE ε4 allele is not associated with REM sleep behavior disorder, Neurobiology of Aging (2016), doi: 10.1016/j.neurobiolaging.2016.10.002.

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The dementia-associated APOE εεεε4 allele is not associated with REM sleep behavior disorder

Ziv Gan-Or, MD, PhD,a,b,c, Jacques Y. Montplaisir, MD, PhD,d,e, Jay P. Ross, BSc,b Judes Poirier, PhD

f,g,

Simon C. Warby, PhD,4,5, Isabelle Arnulf, MD, PhD,h, Stephanie Strong, BSc,a, Yves Dauvilliers,

MD, PhD,i, Claire S. Leblond, PhD,1,b, Michele T.M. Hu,MBBS, FRCP, PhD,j,k, Birgit Högl, MD,l,

Ambra Stefani, MD,l, Christelle Charley Monaca, MD, PhD,m, Valérie Cochen De Cock, MD, PhD,n,o,

Michel Boivin, PhD,p,q,Luigi Ferini-Strambi, MD, PhD,r, Giuseppe Plazzi, MD, PhD,s,t, Elena Antelmi,

MD,s, Peter Young, MD,u, Anna Heidbreder, MD,u,Thomas R Barber MA, MBBS, MRCP,j,k, Samuel G.

Evetts BSc Hons, MSc,j,k, Michal Rolinski, BM BCh, BA Hons, MRCP,j,k, Patrick A. Dion, PhD,a,c,

Alex Desautels, MD, PhD,d,v, Jean-François Gagnon, PhD,d,w, Nicolas Dupré, MD, MSc,x, Ronald B.

Postuma, MD, MSc,c,y, and Guy A. Rouleau, MD, PhD,a,b,c.

Affiliations : a

Montreal Neurological Institute, McGill University, Montréal, QC,H3A 0G4, Canada, bDepartment of

Human Genetics, McGill University, H3A 0G4, Montréal, QC, Canada, cDepartment of Neurology and

neurosurgery, McGill University, Montréal, QC, H3A 0G4, Canada, dCentre d’Études Avancées en

Médecine du Sommeil, Hôpital du Sacré-Cœur de Montréal, Montréal, QC, H4J 1C5, Canada,

e

Department of Psychiatry, Université de Montréal, Montréal, QC, H3T 1J4, Canada, fDepartment of

Psychiatry, McGill University, Montréal, QC, H3A 0G4, Canada, gDouglas Mental Health University

Institute, Montréal, QC, H4H 1R3, Canada, hSleep Disorders Unit, Pitié Salpêtrière Hospital, Centre de

Recherche de l’Institut du Cerveau et de la Moelle Epinière and Sorbonne Universities, UPMC Paris 6

univ, Paris, 75013, France, iSleep Unit, National Reference Network for Narcolepsy, Department of

Neurology Hôpital-Gui-de Chauliac, CHU Montpellier, INSERM U1061, Montpellier, 34000, France, j

Oxford Parkinson’s Disease Centre (OPDC), University of Oxford, Oxford, OX1 2JD, United

Kingdom, kNuffield Department of Clinical Neurosciences, University of Oxford, Oxford, OX1 2JD,

United Kingdom, lSleep Disorders Clinic, Department of Neurology, Medical University of Innsbruck,

Innsbruck,6020, Austria, mUniversity Lille north of France, Department of clinical neurophysiology and

sleep center, CHU Lille, Lille, 59000, France, nSleep and neurology unit, Beau Soleil Clinic,

Montpellier, 34070, France, oEuroMov, University of Montpellier, Montpellier, 34095, France, pGRIP,

École de psychologie, Université Laval, Québec city, QC, G1V 0A6, Canada, qInstitute of Genetic,

Neurobiological and Social Foundations of Child Development, Tomsk State University, Tomsk,

634050, Russia, rDepartment of Neurological Sciences, Università Vita-Salute San Raffaele, Milan,

20132, Italy, sDepartment of Biomedical and Neuromotor Sciences (DIBINEM), Alma Mater

Studiorum, University of Bologna, Bologna,40126, Italy, tIRCCS, Institute of Neurological Sciences of

Bologna, Bologna, 40139, Italy, uDepartment of Sleep Medicine and Neuromuscular Disorders,

University of Muenster, 48149, Germany, vDepartment of Neurosciences, Université de Montréal,

Montréal, H3T 1J4, Canada, wDépartement de psychologie, Université du Québec à Montréal, Montréal,

QC, H2L 2C4, Canada, xFaculté de Médecine, Université Laval, CHU de Québec (Enfant-Jésus),

Québec, QC, G1J 1Z4, Canada, yDepartment of Neurology, Montreal General Hospital, Montréal, QC,

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Montreal Neurological Institute, McGill University 1033 Pine Avenue, West,

Ludmer Pavilion, room 327 Montreal, QC, H3A 1A1 Phone: +1-514-398-6821

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Abstract: The current study aimed to examine whether the APOE ε4 allele, associated with dementia with Lewy bodies (DLB), and possibly with dementia in Parkinson’s disease (PD), is also associated with idiopathic REM sleep behavior disorder (RBD). Two SNPs, rs429358 and rs7412, were genotyped

in RBD patients (n=480) and in controls (n=823). APOE ε4 allele frequency was 0.14 among RBD

patients and 0.13 among controls (OR=1.11, 95% CI 0.88-1.40, p=0.41). APOE ε4 allele frequencies

were similar in those who converted to DLB (0.14) and those who converted to PD (0.12) or multiple

system atrophy (0.14, p=1.0). The APOE ε4 allele is neither a risk factor for RBD nor it is associated

with conversion from RBD to DLB or other synucleinopathies.

1. Introduction: Rapid eye movement (REM) sleep behavior disorder (RBD) is currently the strongest

clinical prodromal feature preceding the development of an overt synucleinopathy, including Parkinson’s disease (PD), dementia with Lewy bodies (DLB) or multiple system atrophy (MSA) (Iranzo, et al., 2014). One of the strongest genetic factors associated with DLB is the APOE epsilon4

(ε4) allele(Pickering-Brown, et al., 1994), and PD patients who carry this allele may be at increased risk

for developing dementia. Since both RBD and the APOE ε4 allele are possibly associated with DLB,

and with dementia in PD patients, we aimed to examine whether the APOE ε4 allele is associated with

RBD and conversion to DLB. See Supplementary file for detailed introduction and full list of references.

2. Methods: The study population included idiopathic RBD patients (n=480) and controls (n=823) of

European ancestry. RBD patients were diagnosed using clinical interview and polysomnography according to the ICSD-2 (International Classification of Sleep Disorders, version 2) criteria. The control group was composed of 253 elderly controls (age 59.5±9.8 years, matched to the available age at onset (AAO) of RBD, n=307, age 59.2±11.5), 510 young controls (age 34.0±6.5 years), and additional 60 controls with no available data on age. All control groups had nearly identical frequencies of the APOE

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individuals signed informed consent forms at enrollment, and the study protocols were approved by the respective institutional review boards. DNA was extracted using a standard salting-out protocol. Two single nucleotide polymorphisms (SNPs), rs429358 and rs7412, were genotyped using TaqMan SNP genotyping assays. Genotypes were called using the QuantStudio™ 7 Flex Real-Time PCR System and Software (v 1.0). Goodness of fit test with one degree of freedom was applied to look for deviation from the Hardy-Weinberg equilibrium (HWE) among the controls. Differences in APOE allele or carriage frequencies were analyzed using the Fisher’s exact test, and differences in continuous variables were analyzed using t-test. A logistic regression model with age and sex as covariates was also applied. All statistical analysis was done using SPSS statistics V.23 (IBM Inc.). Detailed methods can be found in the supplementary file.

3. Results: The allele frequency of APOE ε4 was 0.14 among RBD patients and 0.13 among controls

(OR=1.11, 95% CI 0.88-1.40, p=0.41). Overall, 25.8% of RBD patients carried at least one APOE ε4

compared to 23.0% among controls (p=0.25, Fisher’s exact test), and there were more homozygous

carriers of the APOE ε4 allele among controls (3.2%) as compared to RBD patients (2.7%). Logistic

regression model adjusted for age and sex also demonstrated lack of association between APOE ε4

allele carriage and risk for RBD (OR = 1.25, 95% CI 0.87-1.79, p=0.23). There was no difference in

AAO when comparing carriers (n=88) and non-carriers (n=219) of the APOE ε4 allele (59.1 ± 8.4 vs.

59.3 ± 12.6 years, respectively, p=0.92, t-test). A total of 140 RBD patients (29.2%) were reported to have converted to either PD (n=98, 70% of the converters), dementia/DLB (n=28, 20%) or MSA (n=14,

10%). The carrier frequencies of one or more APOE ε4 in these groups were similar; 23.5%, 25.0% and

28.6%, respectively (p=0.91), and the allele frequencies were 0.12, 0.14 and 0.14 (p=1.0). The APOE ε4

allele frequency among those that did not convert was slightly higher, 0.15 (Table 1), with a total of

26.5% carriers of at least one APOE ε4 allele, compared to 24.3% among those who converted (p=0.65).

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4. Discussion: Although RBD is a strong risk factor for developing DLB, and although DLB was

reported to be associated with the APOE ε4 allele, our results demonstrate lack of association between

the APOE ε4 allele and RBD or its age at onset. These and previous results further suggest that RBD

may have a distinct genetic background; it is associated with GBA mutations(Gan-Or, et al., 2015b), but

unlike PD it is not associated with LRRK2 mutations(Fernandez-Santiago, et al., 2016), and unlike DLB

it is not associated with the APOE ε4 allele. Thus far, GBA, SCARB2, and potentially SNCA (Gan-Or, et

al., 2015a) overlap between RBD, PD and DLB (Supplementary Figure 1, see Supplementary file). Whether RBD has additional, unique genetic factors that were not identified in PD or DLB cohorts is

still to be determined. Our current study identified similar frequencies of APOE ε4 allele in those who

progressed to PD, DLB and MSA, suggesting that APOE alleles do not affect the type of subsequent synucleinopathy. Our study has some limitations, and a more detailed discussion including full list of references can be found in the supplementary file. Our results support a distinct genetic background for RBD-associated neurodegeneration, probably suggesting a specific genetic association with synucleinopathy rather than tauopathy/amyloidopathy.

References:

Fernandez-Santiago, R., Iranzo, A., Gaig, C., Serradell, M., Fernandez, M., Tolosa, E., Santamaria, J., Ezquerra, M. 2016. Absence of LRRK2 mutations in a cohort of patients with idiopathic REM sleep behavior disorder. Neurology 86(11), 1072-3. doi:10.1212/WNL.0000000000002304.

Gan-Or, Z., Girard, S.L., Noreau, A., Leblond, C.S., Gagnon, J.F., Arnulf, I., Mirarchi, C., Dauvilliers, Y., Desautels, A., Mitterling, T., Cochen De Cock, V., Frauscher, B., Monaca, C., Hogl, B., Dion, P.A., Postuma, R.B., Montplaisir, J.Y., Rouleau, G.A. 2015a. Parkinson's Disease Genetic Loci in Rapid Eye Movement Sleep Behavior Disorder. J Mol Neurosci 56(3), 617-22. doi:10.1007/s12031-015-0569-7.

Gan-Or, Z., Mirelman, A., Postuma, R.B., Arnulf, I., Bar-Shira, A., Dauvilliers, Y., Desautels, A., Gagnon, J.F., Leblond, C.S., Frauscher, B., Alcalay, R.N., Saunders-Pullman, R., Bressman, S.B., Marder, K., Monaca, C., Hogl, B., Orr-Urtreger, A., Dion, P.A., Montplaisir, J.Y., Giladi, N., Rouleau, G.A. 2015b. GBA mutations are associated with Rapid Eye Movement Sleep Behavior Disorder. Ann Clin Transl Neurol 2(9), 941-5. doi:10.1002/acn3.228.

Iranzo, A., Fernandez-Arcos, A., Tolosa, E., Serradell, M., Molinuevo, J.L., Valldeoriola, F., Gelpi, E., Vilaseca, I., Sanchez-Valle, R., Llado, A., Gaig, C., Santamaria, J. 2014. Neurodegenerative disorder risk in idiopathic REM sleep behavior disorder: study in 174 patients. PLoS One 9(2), e89741.

doi:10.1371/journal.pone.0089741.

Pickering-Brown, S.M., Mann, D.M., Bourke, J.P., Roberts, D.A., Balderson, D., Burns, A., Byrne, J., Owen, F. 1994. Apolipoprotein E4 and Alzheimer's disease pathology in Lewy body disease and in other beta-amyloid-forming diseases. Lancet 343(8906), 1155.

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ZGO received consultation fees from Sanofi/Genzyme. JYM reports grants from Merck, GlaxoSmithKline, received speaking honoraria from Valeant Pharmaceutical, and Otsuka Pharmaceutical, serves on the advisory boards of Sanofi-Aventis, Servier, Merck, Jazz Pharma, Valeant Pharma, Impax Laboratories, Glaxo-SmithKline, UCB Canada, received consultancy fees from Otsuka Pharma, and Valeant Pharma. JPR reports no conflict of interests. JP reports no conflict of interests. SCW received honoraria from Pfizer, Bristol-Myers Squibb, SmithKline Beecham and Eli Lilly. IA received speaker honoraria form UCB Pharma. SS reports no conflict of interests. YD is on the advisory board and received travel and consultancy fees from UCB Phrma, bioprojet, and Jazz Pharma. CSL reports no conflict of interests. MTH reports no conflict of interests. BH received grant from UCB, speaker honoraria from UCB, Otsuka, Abbvie, Lundbeck, Lilly, Mundipharma. Serving on advisory boards or consulting for Mundipharma, Axovant. Received travel support from Habel Medizintechnik, Vivisol. AS reports no conflict of interests. CCM received fees for serving on advisory board of UCB pharma, lecture fees from UCB Pharma, Orkyn. VCD received funding from Orkyn, LVL medical, Teva and UCB. MB reports no conflict of interests. LFS reports no conflict of interests. GP served on the advisory board of UCB pharma, Jazz pharmaceuticals and Bioproject. EA reports no conflict of interests. PY received honoraria for speakers bureaus by Sanofi Genzyme, Biomarin, UCB pharma, Medice, ResMed and Heinen und Loewenstein. Member of advisory boards for Sanofi Genzyme, Biomarin, Vanda and Medice. AH received travel support Habel Medizintechnik, received lecture honoraria from UCB, Heinen und Löwenstein. TRB reports no conflict of interests. SGE reports no conflict of interests. MR reports no conflict of interests. PAD reports no conflict of interests. AD received research grants from Novartis pharma, Jazz Pharmaceuticals, Biron soins du sommeil. Received speaker honoraria from UCB and Paladin labs.

Acknowledgements

We would like to thank all the participants in the study. This work was funded by a grant to ZGO from the Michael J. Fox Foundation for Parkinson’s research. Part of this work was funded by an interface grant to IA from INSERM. The French DNA collection was promoted by the Association pour le Développement et l'Organisation de la Recherche en Pneumologie et sur le Sommeil (ADOREPS), project PARAGEN, PI Isabelle Arnulf. The Oxford Discovery cohort was funded by the Monument Trust Discovery Award from Parkinson’s UK and supported by the National Institute for Health Research (NIHR), Oxford Biomedical Research Centre based at Oxford University Hospitals, NHS Trust, University of Oxford, and the Dementias and Neurodegenerative Diseases Research Network (DeNDRoN). Part of this work was funded by the Weston Brain Institute (grants to JYM and JP) and the J.L. Levesque Foundation (grant to JP). Part of this work was funded by grants to PY from the Lowensteinstiftung and the German Ministry of Education and Science (BMBF). ZGO is supported by a postdoctoral fellowship from the CIHR. JFG holds a Canada Research Chair on Cognitive Decline in Pathological Aging. GAR holds a Canada Research Chair in Genetics of the Nervous System and the Wilder Penfield Chair in Neurosciences. We thank Cynthia Bourassa, Sandra Laurent, Helene Catoire, Pascale Hince and Vessela Zaharieva for their assistance.

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Table 1. APOE haplotypes in individuals with RBD and controls

APOE εεεε2/εεεε2 n, (%) εεεε2/εεεε3 n, (%) εεεε3/εεεε3 n, (%) εεεε2/εεεε4 n, (%) εεεε3/εεεε4 n, (%) εεεε4/εεεε4 n, (%) Total carriers of εεεε4, n (%) εεεε4 allele frequency RBD patients, n=480 4 (0.8) 51 (10.6) 301 (62.7) 4 (0.8) 107 (22.3) 13 (2.7) 124 (25.8) 0.14 RBD converted to synucleinopathya, n=140 3 (2.1) 12 (8.6) 91 (65.0) 1 (0.7) 32 (22.9) 1 (0.7) 34 (24.3) 0.13 RBD not converted to synucleinopathy, n=340 1 (0.3) 39 (11.5) 210 (61.8) 3 (0.9) 75 (22.1) 12 (3.5) 90 (26.5) 0.15 Controls, n=823 5 (0.6) 111 (13.5) 518 (62.9) 14 (1.7) 149 (18.1) 26 (3.2) 189 (23.0) 0.13 n, number; RBD, REM sleep behavior disorder

a

1

The dementia-associated APOE 4 allele is not associated with REM sleep behavior disorder

Ziv Gan-Or, MD, PhD,a,b,c, Jacques Y. Montplaisir, MD, PhD,d,e, Jay P. Ross, BSc,b Judes Poirier, PhD f,g, Simon C. Warby, PhD,4,5, Isabelle Arnulf, MD, PhD,h, Stephanie Strong, BSc,a, Yves Dauvilliers, MD, PhD,i, Claire S. Leblond, PhD,1,b, Michele T.M. Hu,MBBS, FRCP, PhD,j,k, Birgit Högl, MD,l, Ambra Stefani, MD,l, Christelle Charley Monaca, MD, PhD,m, Valérie Cochen De Cock, MD, PhD,n,o, Michel Boivin, PhD,p,q,Luigi Ferini-Strambi, MD, PhD,r,

Giuseppe Plazzi, MD, PhD,s,t, Elena Antelmi, MD,s, Peter Young, MD,u, Anna Heidbreder, MD,u, Thomas R Barber MA, MBBS, MRCP,j,k, Samuel G. Evetts BSc Hons, MSc,j,k, Michal Rolinski, BM BCh, BA Hons, MRCP,j,k, Patrick A. Dion, PhD,a,c, Alex Desautels, MD, PhD,d,v, Jean-François Gagnon, PhD,d,w, Nicolas Dupré, MD, MSc,x, Ronald B. Postuma, MD, MSc,c,y, and Guy A. Rouleau, MD, PhD,a,b,c.

Affiliations :

a_{Montreal Neurological Institute, McGill University, Montréal, QC, H3A 0G4, Canada,} b_{Department of Human Genetics, McGill University, H3A 0G4, Montréal, QC, Canada,} c_{Department of Neurology and neurosurgery, McGill University, Montréal, QC, H3A 0G4,}

Canada, dCentre d’Études Avancées en Médecine du Sommeil, Hôpital du Sacré-Cœur de Montréal, Montréal, QC,H4J 1C5, Canada, eDepartment of Psychiatry, Université de Montréal, Montréal, QC, H3T 1J4, Canada, f_{Department of Psychiatry, McGill University, Montréal, QC,}

H3A 0G4, Canada, gDouglas Mental Health University Institute, Montréal, QC, H4H 1R3, Canada,

h_{Sleep Disorders Unit, Pitié Salpêtrière Hospital, Centre de Recherche de l’Institut du Cerveau et}

de la Moelle Epinière and Sorbonne Universities, UPMC Paris 6 univ, Paris,75013, France, i_Sleep

Unit, National Reference Network for Narcolepsy, Department of Neurology Hôpital-Gui-de Chauliac, CHU Montpellier, INSERM U1061, Montpellier, 34000, France, jOxford Parkinson’s Disease Centre (OPDC), University of Oxford, Oxford, OX1 2JD, United Kingdom, k_Nuffield

Department of Clinical Neurosciences, University of Oxford, Oxford, OX1 2JD, United Kingdom,

l_{Sleep Disorders Clinic, Department of Neurology, Medical University of Innsbruck, Innsbruck,}

6020, Austria, mUniversity Lille north of France, Department of clinical neurophysiology and sleep center, CHU Lille, Lille, 59000, France, nSleep and neurology unit, Beau Soleil Clinic, Montpellier, 34070, France, oEuroMov, University of Montpellier, Montpellier, 34095, France,

p_{GRIP, École de psychologie, Université Laval, Québec city, QC, G1V 0A6, Canada,} q_{Institute of}

Genetic, Neurobiological and Social Foundations of Child Development, Tomsk State University, Tomsk, 634050, Russia, rDepartment of Neurological Sciences, Università Vita-Salute San Raffaele, Milan, 20132, Italy, s_{Department of Biomedical and Neuromotor Sciences (DIBINEM),}

Alma Mater Studiorum, University of Bologna, Bologna, 40126, Italy, tIRCCS, Institute of Neurological Sciences of Bologna, Bologna, 40139, Italy, uDepartment of Sleep Medicine and Neuromuscular Disorders, University of Muenster, 48149, Germany, v_{Department of}

Neurosciences, Université de Montréal, Montréal, H3T 1J4, Canada, wDépartement de psychologie, Université du Québec à Montréal, Montréal, QC, H2L 2C4, Canada, xFaculté de

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Médecine, Université Laval, CHU de Québec (Enfant-Jésus), Québec, QC, G1J 1Z4, Canada,

y_{Department of Neurology, Montreal General Hospital, Montréal, QC, H3G 1A4, Canada.}

Correspondence:

Ziv Gan-Or

Montreal Neurological Institute, McGill University 1033 Pine Avenue, West,

Ludmer Pavilion, room 327 Montreal, QC, H3A 1A1 Phone: +1-514-398-6821

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Abstract

A significant proportion of individuals with REM sleep behavior disorder (RBD) will progress to

dementia with Lewy bodies (DLB) and Parkinson’s disease (PD). We aimed to examine whether

the APOE 4 allele, associated with DLB, and possibly with dementia in PD, is also associated

with idiopathic RBD. The two SNPs tagging the different APOE alleles (rs429358 and rs7412)

were genotyped in individuals who were initially diagnosed with RBD (n=480) and in controls

(n=823). APOE 4 allele frequency was 0.14 among RBD patients and 0.13 among controls

(OR=1.11, 95% CI 0.88-1.40, p=0.41), and this lack of association remained after adjustment for

age and sex. Furthermore, allele frequencies of APOE 4 were similar in those who converted to

DLB (0.14) and those who converted to PD (0.12) or multiple system atrophy (0.14, p=1.0). The

APOE 4 allele is neither a risk factor for RBD nor it is associated with conversion from RBD to

DLB or other synucleinopathies.

4

Introduction

Rapid eye movement (REM) sleep behavior disorder (RBD), characterized by lack of muscle

atonia and enacting of dreams during REM sleep, is currently the strongest clinical prodromal

feature preceding the development of an overt synucleinopathy. With long term follow-up, more

than 80% of individuals with idiopathic RBD developed either Parkinson’s disease (PD), dementia

with Lewy bodies (DLB) or multiple system atrophy (MSA) (Iranzo, et al., 2014,Schenck, et al.,

2013). It was suggested that RBD may define a subtype of PD patients (Fereshtehnejad, et al.,

2015,Gagnon, et al., 2004) with cognitive decline (Gagnon, et al., 2009,Vendette, et al., 2007),

dementia (Anang, et al., 2014), hallucinations (Sixel-Doring, et al., 2011) and autonomic

dysfunction (Postuma, et al., 2008), as compared to PD patients without RBD. In addition,

pathological studies in brains of PD patients with and without RBD demonstrated a more

widespread -synuclein accumulation in those associated with RBD (Postuma, et al., 2015a).

If indeed RBD represents a subtype of PD, or a subtype of synucleinopathy, it is possible

that it has specific genetic background. A preliminary study that examined the association of RBD

with several genetic risk factors for PD identified an association mainly with MAPT and SCARB2,

and marginal or lack of association with other markers (Gan-Or, et al., 2015a). A recent study

suggested that RBD is associated with mutations in GBA in both idiopathic RBD and PD cohorts

(Gan-Or, et al., 2015b). This association was stronger than the association of GBA mutations with

PD in a similar population (Noreau, et al., 2011), suggesting that GBA may be one of the genetic

factors that is more specific to RBD. Furthermore, the association of GBA mutations with DLB

(Nalls, et al., 2013) also seems to be stronger than the association with PD (Sidransky, et al., 2009).

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2016,Pont-Sunyer, et al., 2015,Saunders-Pullman, et al., 2015), further supporting the hypothesis

that RBD has a distinct genetic background.

One of the strongest genetic factors associated with DLB is the APOE epsilon4 (allele

(Pickering-Brown, et al., 1994), and PD patients who carry this allele may be at increased risk for

developing dementia (Pankratz, et al., 2006), although negative results were also reported for

these associations (Jasinska-Myga, et al., 2007,Lovati, et al., 2010). Since both RBD and the APOE

 allele are possibly associated with DLB, and with dementia in PD patients, we aimed to examine whether the APOE 4 allele is associated with RBD and conversion to DLB.

Methods

Population

The study population included consecutively recruited, unrelated idiopathic RBD patients (n=480)

and controls (n=823) of European ancestry. RBD patients were collected by collaborators from the

international RBD study group and were diagnosed using clinical interview and polysomnography

according to the ICSD-2 (International Classification of Sleep Disorders, version 2)

criteria.(Thorpy, 2012) The control group was composed of 253 elderly controls (age 59.5±9.8

years, matched to the available age at onset (AAO) of RBD, n=307, age 59.2±11.5), 510 young

controls (age 34.0±6.5 years), and additional 60 controls with no available data on age. However,

all control groups had nearly identical frequencies of the APOE 4 allele (0.13, 0.13 and 0.14,

respectively), suggesting lack of age-effect, which allowed us to analyze all controls combined.

All individuals signed informed consent forms at enrollment, and the study protocols were

6

Genotyping

DNA was extracted using a standard salting-out protocol. To determine the APOE haplotypes, two

tagging single nucleotide polymorphisms (SNPs), rs429358 and rs7412, were genotyped using

TaqMan SNP genotyping assays (C___3084793_20 and C____904973_10, respectively,

ThermoFisher Scientific Inc.) according to the manufacturer's instructions. Genotypes were called

using the QuantStudio™ 7 Flex Real-Time PCR System and Software (v 1.0). Carriers of T in rs429358 and T in rs7412 were determined as carriers of the 2 allele, carriers of T in rs429358

and C in rs7412 were determined as carriers of the 3 allele, and carriers of C in rs429358 and C

in rs7412 were determined as carriers of the 4 allele. Of this cohort, the GBA gene was sequenced

in 265 RBD patients (Gan-Or, et al., 2015b), and nine PD-associated SNPs were genotyped in 261

patients(Gan-Or, et al., 2015a).

Statistical analysis

Categorical variables are presented as percentage or frequencies, whereas continuous variables are

presented as mean ± standard deviation. Goodness of fit test with one degree of freedom was

applied to look for deviation from the Hardy-Weinberg equilibrium (HWE) among the controls.

Differences in APOE allele or carriage frequencies were analyzed using the Fisher’s exact test,

and differences in continuous variables were analyzed using t-test. To further avoid a potential

bias due to age, and since sex distribution was different among patients with RBD vs. controls, a

logistic regression model with age and sex as covariates was also applied. All statistical analysis

was done using SPSS statistics V.23 (IBM Inc.).

7

Lack of association between the APOE 4, RBD risk and its age at onset

Table 1 details the different APOE alleles in RBD patients and controls. The frequency of the two

SNPs defining the APOE alleles did not deviate from HWE. The allele frequency of APOE 4 was

0.14 among RBD patients and 0.13 among controls (OR=1.11, 95% CI 0.88-1.40, p=0.41).

Overall, 25.8% of RBD patients carried at least one APOE 4 compared to 23.0% among controls

(p=0.25, Fisher’s exact test), and there were more homozygous carriers of the APOE 4 allele

among controls (3.2%) as compared to RBD patients (2.7%). Logistic regression model adjusted

for age and sex also demonstrated lack of association between APOE 4 allele carriage and risk

for RBD (OR = 1.25, 95% CI 0.87-1.79, p=0.23). Data on age at onset (AAO) of RBD was

available for 307 individuals, and there was no difference in AAO when comparing carriers (n=88)

and non-carriers (n=219) of the APOE 4 allele (59.1 ± 8.4 vs. 59.3 ± 12.6 years, respectively,

p=0.92, t-test).

The APOE 4 allele is not associated with conversion to PD, DLB or MSA.

Since most of the patients in our cohort are being followed-up longitudinally, we examined

whether the APOE 4 allele is associated with conversion to either PD, dementia/DLB, or MSA.

A total of 140 RBD patients (29.2%) were reported to have converted to either PD (n=98, 70% of

the converters), dementia/DLB (n=28, 20%) or MSA (n=14, 10%). The carrier frequencies of one

or more APOE 4 in these groups were similar; 23.5%, 25.0% and 28.6%, respectively (p=0.91),

and the allele frequencies were 0.12, 0.14 and 0.14 (p=1.0). The APOE 4 allele frequency among

those that did not convert was slightly higher, 0.15 (Table 1), with a total of 26.5% carriers of at

8

Discussion

Although RBD is a strong risk factor for developing DLB (Iranzo, et al., 2014,Postuma, et al.,

2015b). and although DLB was reported to be associated with the APOE 4 allele (Kobayashi, et

al., 2011,Lane, et al., 2009,Pickering-Brown, et al., 1994), our results demonstrate lack of

association between the APOE 4 allele and RBD or its age at onset. These and previous results

(Fernandez-Santiago, et al., 2016,Gan-Or, et al., 2015a,Gan-Or, et al., 2015b,Saunders-Pullman,

et al., 2015) further suggest that RBD may have a distinct genetic background; it is associated with

GBA mutations (Gan-Or, et al., 2015b), but unlike PD it is not associated with LRRK2 mutations

(Fernandez-Santiago, et al., 2016,Saunders-Pullman, et al., 2015), and unlike DLB it is not

associated with the APOE 4 allele. Thus far, GBA, SCARB2, and potentially SNCA overlap

between RBD, PD and DLB (Figure 1) (Bras, et al., 2014,Gan-Or, et al., 2015a,Gan-Or, et al.,

2015b). Whether RBD has additional, unique genetic factors that were not identified in PD or DLB

cohorts is still to be determined.

Since PD patients with RBD are likely to develop dementia and hallucinations (Anang, et

al., 2014,Sinforiani, et al., 2008), eventually presenting a phenotype similar to DLB, and based on

the current and previous genetic and post-mortem results (Postuma, et al., 2015a), we hypothesize

that RBD-associated synucleinopathy (the central common area in Figure 1) is the same

clinical-pathological entity, whether it is defined as parkinsonism first with subsequent dementia and

hallucinations, or whether it is defined as DLB with subsequent parkinsonism. In that sense, RBD

can be considered as a marker for diffuse synucleinopathy, which may be a better description of

9

also demonstrated a more diffuse synucleinopathy (Choi, et al., 2011,Neumann, et al., 2009,Wong,

et al., 2004), further supporting this notion. However, others suggest that PD and DLB should

remain separate entities, at least until better genetic, molecular and clinical data will allow better

definitions of these diseases and their potential subgroups (Boeve, et al., 2016). It is possible that

stochastic events, or other genetic or environmental factors, determine whether -synuclein will

first be deposited in brain areas associated with dementia and later in the areas associated with

parkinsonism, or vice versa (Lai, et al., 2008,Lai and Siegel, 2003). The observation that

-synuclein can spread in the brain in a prion-like fashion (Bernis, et al., 2015,Danzer, et al.,

2009,Freundt, et al., 2012) may support a stochastic progression hypothesis, yet additional

neuro-pathological studies are needed to examine this possibility. Therefore, there are two possible

explanations for the lack of association between the APOE 4 allele and conversion to DLB in our

cohort. First, as previously suggested (Bras, et al., 2014), it is possible that the association of the

APOE 4 allele with DLB is due to the component of DLB patients who also have a tauopathy,

and that the association of RBD with DLB is with those who have more pure synucleinopathy.

Alternatively, since the majority of our cohort had not yet converted to an overt synucleinopathy,

it is possible that once a larger number would convert, an association between APOE 4 allele and

conversion to DLB may arise. Hence, a follow-up study will be needed to determine this

possibility.

The association of RBD with the more devastating synucleinopathy, MSA, also

necessitates more study. Whether unique genetic or environmental factors affect the risk to

progress from RBD to MSA is still unknown. Our current study identified similar frequencies of

APOE 4 allele in those who progressed to PD, DLB and MSA, suggesting that APOE is not one

10

MSA as well,(Mitsui, et al., 2015) however this observation is awaiting replications in additional

cohorts.

Our study has some limitations. The control population was younger than the RBD group.

To tackle this limitation, we took two approaches. First, we demonstrated that the frequencies of

the APOE 4 allele were similar across generations (i.e. in the elderly and young control groups),

which rules out a potential bias. Furthermore, we also performed a logistic regression model with

adjustment for age, which further demonstrated lack of association between the APOE 4 allele

and RBD. Another possible limitation would stem from RBD patients being recruited in multiple

centers, which could have led to a potential population dependent bias if some cohorts are enriched

in APOE 4 allele carriers. However, since the frequencies of the APOE 4 allele were similar

across centers, this possibility was ruled out.

To conclude, our results support a distinct genetic background for RBD-associated

neurodegeneration, probably suggesting a specific genetic association with synucleinopathy rather

than tauopathy/amyloidopathy. To examine the hypotheses raised by the current and previous

work, larger studies are necessary, including genome wide association and next-generation

11

Acknowledgements

We would like to thank all the participants in the study. This work was funded by a grant to ZGO from the Michael J. Fox Foundation for Parkinson’s research. Part of this work was funded by an interface grant to IA from INSERM. The French DNA collection was promoted by the Association pour le Développement et l'Organisation de la Recherche en Pneumologie et sur le Sommeil (ADOREPS), project PARAGEN, PI Isabelle Arnulf. The Oxford Discovery cohort was funded by the Monument Trust Discovery Award from Parkinson’s UK and supported by the National Institute for Health Research (NIHR), Oxford Biomedical Research Centre based at Oxford University Hospitals, NHS Trust, University of Oxford, and the Dementias and Neurodegenerative Diseases Research Network (DeNDRoN). Part of this work was funded by the Weston Brain Institute (grants to JYM and JP) and the J.L. Levesque Foundation (grant to JP). Part of this work was funded by grants to PY from the Lowensteinstiftung and the German Ministry of Education and Science (BMBF). ZGO is supported by a postdoctoral fellowship from the CIHR. JFG holds a Canada Research Chair on Cognitive Decline in Pathological Aging. GAR holds a Canada Research Chair in Genetics of the Nervous System and the Wilder Penfield Chair in Neurosciences. We thank Cynthia Bourassa, Sandra Laurent, Helene Catoire, Pascale Hince and Vessela Zaharieva for their assistance.

Financial Disclosures

ZGO received consultation fees from Sanofi/Genzyme. JYM reports grants from Merck, GlaxoSmithKline, received speaking honoraria from Valeant Pharmaceutical, and Otsuka Pharmaceutical, serves on the advisory boards of Sanofi-Aventis, Servier, Merck, Jazz Pharma, Valeant Pharma, Impax Laboratories, Glaxo-SmithKline, UCB Canada, received consultancy fees from Otsuka Pharma, and Valeant Pharma. JPR reports no conflict of interests. JP reports no conflict of interests. SCW received honoraria from Pfizer, Bristol-Myers Squibb, SmithKline Beecham and Eli Lilly. IA received speaker honoraria form UCB Pharma. SS reports no conflict of interests. YD is on the advisory board and received travel and consultancy fees from UCB Phrma, bioprojet, and Jazz Pharma. CSL reports no conflict of interests. MTH reports no conflict of interests. BH received grant from UCB, speaker honoraria from UCB, Otsuka, Abbvie, Lundbeck, Lilly, Mundipharma. Serving on advisory boards or consulting for Mundipharma, Axovant. Received travel support from Habel Medizintechnik, Vivisol. AS reports no conflict of interests. CCM received fees for serving on advisory board of UCB pharma, lecture fees from UCB Pharma, Orkyn. VCD received funding from Orkyn, LVL medical, Teva and UCB. MB reports no conflict of interests. LFS reports no conflict of interests. GP served on the advisory board of UCB pharma, Jazz pharmaceuticals and Bioproject. EA reports no conflict of interests. PY received honoraria for speakers bureaus by Sanofi Genzyme, Biomarin, UCB pharma, Medice, ResMed and Heinen und Loewenstein. Member of advisory boards for Sanofi Genzyme, Biomarin, Vanda and Medice. AH received travel support Habel Medizintechnik, received lecture honoraria from UCB, Heinen und Löwenstein. TRB reports no conflict of interests. SGE reports no conflict of interests. MR reports no conflict of interests. PAD reports no conflict of interests. AD received

12

research grants from Novartis pharma, Jazz Pharmaceuticals, Biron soins du sommeil. Received speaker honoraria from UCB and Paladin labs.

13

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Figure 1. Venn diagram of the genetic overlap between PD, DLB and RBD.

While GBA mutations, SCARB2 and possibly SNCA variants are associated with all three

conditions, other genetic variants such as APOE 4 in DLB and LRRK2 mutations in PD are

distinctively associated with each condition but not with RBD. It is therefore likely that

RBD-associated neurodegeneration (the overlapping area of PD, DLB and RBD) may have distinct

genetic background. While thus far no genetic variants that are uniquely associated with RBD were

discovered, it is possible that such genetic risk factors do exist, and that they were not discovered