title: Targeted NGS gene panel identifies mutations in RSPH1 causing primary ciliary dyskinesia and a common mechanism for ciliary central pair agenesis due to radial spoke defects.
abstract: Primary ciliary dyskinesia (PCD) is an inherited chronic respiratory obstructive disease with randomized body laterality and infertility, resulting from cilia and sperm dysmotility. PCD is characterized by clinical variability and extensive genetic heterogeneity, associated with different cilia ultrastructural defects and mutations identified in >20 genes. Next generation sequencing (NGS) technologies therefore present a promising approach for genetic diagnosis which is not yet in routine use. We developed a targeted panel-based NGS pipeline to identify mutations by sequencing of selected candidate genes in 70 genetically undefined PCD patients. This detected loss-of-function RSPH1 mutations in four individuals with isolated central pair (CP) agenesis and normal body laterality, from two unrelated families. Ultrastructural analysis in RSPH1-mutated cilia revealed transposition of peripheral outer microtubules into the 'empty' CP space, accompanied by a distinctive intermittent loss of the central pair microtubules. We find that mutations in RSPH1, RSPH4A and RSPH9, which all encode homologs of components of the 'head' structure of ciliary radial spoke complexes identified in Chlamydomonas, cause clinical phenotypes that appear to be indistinguishable except at the gene level. By high-resolution immunofluorescence we identified a loss of RSPH4A and RSPH9 along with RSPH1 from RSPH1-mutated cilia, suggesting RSPH1 mutations may result in loss of the entire spoke head structure. CP loss is seen in up to 28% of PCD cases, in whom laterality determination specified by CP-less embryonic node cilia remains undisturbed. We propose this defect could arise from instability or agenesis of the ciliary central microtubules due to loss of their normal radial spoke head tethering.
authors: Molecular Medicine Unit and Birth Defects Research Centre, Institute of Child Health, University College London, London WC1N 1EH, UK Present address: Department of Medical and Molecular Genetics, Division of Genetics and Molecular Medicine, King's College London School of Medicine, Guy's Hospital, London SE1 9RT, UK.
authors: Alexandros
authors: A
authors: Onoufriadis
authors: Department of Paediatric Respiratory Medicine, Royal Brompton and Harefield NHS Trust, Sydney Street, London SW3 6NP, UK.
authors: Amelia
authors: A
authors: Shoemark
authors: Molecular Medicine Unit and Birth Defects Research Centre, Institute of Child Health, University College London, London WC1N 1EH, UK.
authors: Miriam
authors: M
authors: Schmidts
authors: Molecular Medicine Unit and Birth Defects Research Centre, Institute of Child Health, University College London, London WC1N 1EH, UK.
authors: Mitali
authors: M
authors: Patel
authors: Laboratoire Cardiogénétique, Equipe d'Accueil 4173, Université Lyon 1, Hôpital Nord-Ouest, Villefranche sur Saône, Lyon, France Laboratoire Cardiogénétique, Hospices Civils de Lyon, Groupe Hospitalier Est, 69677 Bron, France.
authors: Gina
authors: G
authors: Jimenez
authors: Laboratoire Cardiogénétique, Equipe d'Accueil 4173, Université Lyon 1, Hôpital Nord-Ouest, Villefranche sur Saône, Lyon, France Laboratoire Cardiogénétique, Hospices Civils de Lyon, Groupe Hospitalier Est, 69677 Bron, France.
authors: Hui
authors: H
authors: Liu
authors: Department of Infection, Immunity and Inflammation, Division of Child Health, CSB, University of Leicester, Leicester LE2 7LX, UK.
authors: Biju
authors: B
authors: Thomas
authors: Department of Paediatric Respiratory Medicine, Royal Brompton and Harefield NHS Trust, Sydney Street, London SW3 6NP, UK.
authors: Mellisa
authors: M
authors: Dixon
authors: Department of Infection, Immunity and Inflammation, Division of Child Health, CSB, University of Leicester, Leicester LE2 7LX, UK.
authors: Robert A
authors: RA
authors: Hirst
authors: Department of Infection, Immunity and Inflammation, Division of Child Health, CSB, University of Leicester, Leicester LE2 7LX, UK.
authors: Andrew
authors: A
authors: Rutman
authors: Institute of Ophthalmology, University College London, London EC1V 9EL, UK.
authors: Thomas
authors: T
authors: Burgoyne
authors: Department of Infection, Immunity and Inflammation, Division of Child Health, CSB, University of Leicester, Leicester LE2 7LX, UK.
authors: Christopher
authors: C
authors: Williams
authors: Molecular Medicine Unit and Birth Defects Research Centre, Institute of Child Health, University College London, London WC1N 1EH, UK.
authors: Juliet
authors: J
authors: Scully
authors: Service de Pneumologie, Centre Hospitalier Régional de Roubaix, Hôpital Victor Provo, Roubaix, France.
authors: Florence
authors: F
authors: Bolard
authors: Département de Pneumologie, Centre Hospitalier Régional Universitaire de Lille, Hôpital Albert Calmette, Université Lille 2, Lille, France.
authors: Jean-Jacques
authors: JJ
authors: Lafitte
authors: Molecular Medicine Unit and Birth Defects Research Centre, Institute of Child Health, University College London, London WC1N 1EH, UK.
authors: Philip L
authors: PL
authors: Beales
authors: Department of Paediatric Respiratory Medicine, Royal Brompton and Harefield NHS Trust, Sydney Street, London SW3 6NP, UK.
authors: Claire
authors: C
authors: Hogg
authors: Department of Biology, Marquette University, Milwaukee, WI 53233, USA.
authors: Pinfen
authors: P
authors: Yang
authors: General and Adolescent Paediatric Unit, Institute of Child Health, University College London, London, UK.
authors: Eddie M K
authors: EM
authors: Chung
authors: School of Veterinary Medicine and Science, University of Nottingham, Leicestershire LE12 5RD, UK Advanced Data Analysis Centre, University of Nottingham, Sutton Bonington Campus, Leicestershire LE12 5RD, UK.
authors: Richard D
authors: RD
authors: Emes
authors: Department of Infection, Immunity and Inflammation, Division of Child Health, CSB, University of Leicester, Leicester LE2 7LX, UK Department of Respiratory Medicine, Portex Unit, Institute of Child Health, University College London and Great Ormond Street Hospital, 30 Guilford Street, London WC1N 1EH, UK.
authors: Christopher
authors: C
authors: O'Callaghan
authors: Laboratoire Cardiogénétique, Equipe d'Accueil 4173, Université Lyon 1, Hôpital Nord-Ouest, Villefranche sur Saône, Lyon, France Laboratoire Cardiogénétique, Hospices Civils de Lyon, Groupe Hospitalier Est, 69677 Bron, France Service de Cardiologie Pédiatrique, Hospices Civils de Lyon, Groupe Hospitalier Est, 69677 Bron, France.
authors: Patrice
authors: P
authors: Bouvagnet
authors: Molecular Medicine Unit and Birth Defects Research Centre, Institute of Child Health, University College London, London WC1N 1EH, UK h.mitchison@ucl.ac.uk.
authors: Hannah M
authors: HM
authors: Mitchison
mesh_terms: D054468:Axoneme / Q000378:metabolism / Q000502:physiology; D003598:Cytoskeletal Proteins / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics*; D005260:Female; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D007619:Kartagener Syndrome / Q000235:genetics* / Q000503:physiopathology; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D009154:Mutation; D011506:Proteins / Q000235:genetics
chemical_list: D003598:Cytoskeletal Proteins; D004268:DNA-Binding Proteins; D011506:Proteins; C484746:RSPH1 protein, human; C000589692:RSPH4A protein, human; C573132:RSPH9 protein, human