title: Targeted resequencing and systematic in vivo functional testing identifies rare variants in MEIS1 as significant contributors to restless legs syndrome.
abstract: Restless legs syndrome (RLS) is a common neurologic condition characterized by nocturnal dysesthesias and an urge to move, affecting the legs. RLS is a complex trait, for which genome-wide association studies (GWASs) have identified common susceptibility alleles of modest (OR 1.2-1.7) risk at six genomic loci. Among these, variants in MEIS1 have emerged as the largest risk factors for RLS, suggesting that perturbations in this transcription factor might be causally related to RLS susceptibility. To establish this causality, direction of effect, and total genetic burden of MEIS1, we interrogated 188 case subjects and 182 control subjects for rare alleles not captured by previous GWASs, followed by genotyping of ∼3,000 case subjects and 3,000 control subjects, and concluded with systematic functionalization of all discovered variants using a previously established in vivo model of neurogenesis. We observed a significant excess of rare MEIS1 variants in individuals with RLS. Subsequent assessment of all nonsynonymous variants by in vivo complementation revealed an excess of loss-of-function alleles in individuals with RLS. Strikingly, these alleles compromised the function of the canonical MEIS1 splice isoform but were irrelevant to an isoform known to utilize an alternative 3' sequence. Our data link MEIS1 loss of function to the etiopathology of RLS, highlight how combined sequencing and systematic functional annotation of rare variation at GWAS loci can detect risk burden, and offer a plausible explanation for the specificity of phenotypic expressivity of loss-of-function alleles at a locus broadly necessary for neurogenesis and neurodevelopment.
authors.affiliation: Neurologische Klinik und Poliklinik, Klinikum rechts der Isar, Technische Universität München, 81675 Munich, Germany; Institut für Humangenetik, Helmholtz Zentrum München, 85764 Munich, Germany.
authors.forename: Eva C
authors.initials: EC
authors.lastname: Schulte
authors.affiliation: Center for Human Disease Modeling, Department of Cell Biology, Duke University, Durham, NC 27710, USA.
authors.forename: Maria
authors.initials: M
authors.lastname: Kousi
authors.affiliation: Center for Human Disease Modeling, Department of Cell Biology, Duke University, Durham, NC 27710, USA.
authors.forename: Perciliz L
authors.initials: PL
authors.lastname: Tan
authors.affiliation: Institut für Humangenetik, Helmholtz Zentrum München, 85764 Munich, Germany; Institut für Humangenetik, Technische Universität München, 81675 Munich, Germany.
authors.forename: Erik
authors.initials: E
authors.lastname: Tilch
authors.affiliation: Institut für Humangenetik, Helmholtz Zentrum München, 85764 Munich, Germany.
authors.forename: Franziska
authors.initials: F
authors.lastname: Knauf
authors.affiliation: Institut für Humangenetik, Helmholtz Zentrum München, 85764 Munich, Germany; Institut für Humangenetik, Technische Universität München, 81675 Munich, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Lichtner
authors.affiliation: Paracelsus Elena Klinik, 34128 Kassel, Germany; Klinik für Neurochirurgie, Georg August Universität, 37075 Göttingen, Germany.
authors.forename: Claudia
authors.initials: C
authors.lastname: Trenkwalder
authors.affiliation: Department of Neurology, Medizinische Universität Innsbruck, 6020 Innsbruck, Austria.
authors.forename: Birgit
authors.initials: B
authors.lastname: Högl
authors.affiliation: Department of Neurology, Medizinische Universität Innsbruck, 6020 Innsbruck, Austria.
authors.forename: Birgit
authors.initials: B
authors.lastname: Frauscher
authors.affiliation: Institut für Epidemiologie und Sozialmedizin, Westfälische Wilhelms Universität Münster, 48149 Münster, Germany.
authors.forename: Klaus
authors.initials: K
authors.lastname: Berger
authors.affiliation: Zentrum für Schlafmedizin, Charité Universitätsmedizin, 10117 Berlin, Germany.
authors.forename: Ingo
authors.initials: I
authors.lastname: Fietze
authors.affiliation: Neurologische Klinik und Poliklinik, Klinikum rechts der Isar, Technische Universität München, 81675 Munich, Germany; Interdisziplinäres Schmerzzentrum, Albert-Ludwigs Universität Freiburg, 79106 Freiburg, Germany; Diakoniewerk München-Maxvorstadt, 80799 Munich, Germany.
authors.forename: Magdolna
authors.initials: M
authors.lastname: Hornyak
authors.affiliation: Klinik für Neurologie, Philipps Universität Marburg, 35039 Marburg, Germany.
authors.forename: Wolfgang H
authors.initials: WH
authors.lastname: Oertel
authors.affiliation: Abteilung für Neurologie, Paracelsus Klinikum Osnabrück, 49076 Osnabrück, Germany; Klinische Neurophysiologie, Georg August Universität, 37075 Göttingen, Germany.
authors.forename: Cornelius G
authors.initials: CG
authors.lastname: Bachmann
authors.affiliation: Department of Neurology, Medizinische Universität Wien, 1090 Vienna, Austria.
authors.forename: Alexander
authors.initials: A
authors.lastname: Zimprich
authors.affiliation: Institute of Epidemiology II, Helmholtz Zentrum München, 85764 Munich, Germany.
authors.forename: Annette
authors.initials: A
authors.lastname: Peters
authors.affiliation: Institute of Genetic Epidemiology, Helmholtz Zentrum München, 85764 Munich, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Gieger
authors.affiliation: Institut für Humangenetik, Helmholtz Zentrum München, 85764 Munich, Germany; Institut für Humangenetik, Technische Universität München, 81675 Munich, Germany; Munich Cluster for Systems Neurology (SyNergy), Munich, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Meitinger
authors.affiliation: Munich Cluster for Systems Neurology (SyNergy), Munich, Germany; Max-Planck Institut für Psychiatrie München, 80804 Munich, Germany; Institute of Translational Medicine, University of Liverpool, Liverpool L69 3BX, UK.
authors.forename: Bertram
authors.initials: B
authors.lastname: Müller-Myhsok
authors.affiliation: Center for Human Disease Modeling, Department of Cell Biology, Duke University, Durham, NC 27710, USA.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Katsanis
authors.affiliation: Neurologische Klinik und Poliklinik, Klinikum rechts der Isar, Technische Universität München, 81675 Munich, Germany; Institut für Humangenetik, Helmholtz Zentrum München, 85764 Munich, Germany; Institut für Humangenetik, Technische Universität München, 81675 Munich, Germany; Munich Cluster for Systems Neurology (SyNergy), Munich, Germany; Department of Neurology and Neurosciences, Center for Sleep Sciences and Medicine, Stanford University, Palo Alto, CA 94304, USA. Electronic address: winkelmann@stanford.edu.
authors.forename: Juliane
authors.initials: J
authors.lastname: Winkelmann
mesh_terms: D000818:Animals; D005816:Genetic Complementation Test; D005838:Genotype; D018398:Homeodomain Proteins / Q000235:genetics*; D006801:Humans; D017403:In Situ Hybridization; D013058:Mass Spectrometry; D000075064:Myeloid Ecotropic Viral Integration Site 1 Protein; D009363:Neoplasm Proteins / Q000235:genetics*; D012148:Restless Legs Syndrome / Q000235:genetics*; D015027:Zebrafish / Q000196:embryology
chemical_list: D018398:Homeodomain Proteins; C000616391:MEIS1 protein, human; D000075064:Myeloid Ecotropic Viral Integration Site 1 Protein; D009363:Neoplasm Proteins