Variant-specific changes in RAC3 function disrupt corticogenesis in neurodevelopmental phenotypes

September 14, 2022

Marcello Scala 1 2 3, Masashi Nishikawa 3, Hidenori Ito 3, Hidenori Tabata 3, Tayyaba Khan 4, Andrea Accogli 1, Laura Davids 5, Anna Ruiz 6, Pietro Chiurazzi 7 8, Gabriella Cericola 9, Björn Schulte 10, Kristin G Monaghan 11, Amber Begtrup 11, Annalaura Torella 12 13, Michele Pinelli 12, Anne Sophie Denommé-Pichon 14 15 16, Antonio Vitobello 14 15, Caroline Racine 15 16, Maria Margherita Mancardi 17, Courtney Kiss 18, Andrea Guerin 18, Wendy Wu 4 19, Elisabeth Gabau Vila 20, Bryan C Mak 21, Julian A Martinez-Agosto 21 22 23, Michael B Gorin 21 24 25, Bugrahan Duz 26, Yavuz Bayram 27 28, Claudia M B Carvalho 29 30, Jaime E Vengoechea 5, David Chitayat 31 32 33, Tiong Yang Tan 34, Bert Callewaert 35, Bernd Kruse 9, Lynne M Bird 36 37, Laurence Faivre 14 16, Marcella Zollino 7 8, Saskia Biskup 10 38; Undiagnosed Diseases Network; Telethon Undiagnosed Diseases Program; Pasquale Striano 1 2, Vincenzo Nigro 12 13, Mariasavina Severino 39, Valeria Capra 40, Gregory Costain 4 32 33 41, Koh Ichi Nagata 3 42

Abstract

Variants in RAC3, encoding a small GTPase RAC3 which is critical for the regulation of actin cytoskeleton and intracellular signal transduction, are associated with a rare neurodevelopmental disorder with structural brain anomalies and facial dysmorphism. We investigated a cohort of 10 unrelated participants presenting with global psychomotor delay, hypotonia, behavioural disturbances, stereotyped movements, dysmorphic features, seizures and musculoskeletal abnormalities. MRI of brain revealed a complex pattern of variable brain malformations, including callosal abnormalities, white matter thinning, grey matter heterotopia, polymicrogyria/dysgyria, brainstem anomalies and cerebellar dysplasia. These patients harboured eight distinct de novo RAC3 variants, including six novel variants (NM_005052.3): c.34G > C p.G12R, c.179G > A p.G60D, c.186_188delGGA p.E62del, c.187G > A p.D63N, c.191A > G p.Y64C and c.348G > C p.K116N. We then examined the pathophysiological significance of these novel and previously reported pathogenic variants p.P29L, p.P34R, p.A59G, p.Q61L and p.E62K. In vitro analyses revealed that all tested RAC3 variants were biochemically and biologically active to variable extent, and exhibited a spectrum of different affinities to downstream effectors including p21-activated kinase 1. We then focused on the four variants p.Q61L, p.E62del, p.D63N and p.Y64C in the Switch II region, which is essential for the biochemical activity of small GTPases and also a variation hot spot common to other Rho family genes, RAC1 and CDC42. Acute expression of the four variants in embryonic mouse brain using in utero electroporation caused defects in cortical neuron morphology and migration ending up with cluster formation during corticogenesis. Notably, defective migration by p.E62del, p.D63N and p.Y64C were rescued by a dominant negative version of p21-activated kinase 1. Our results indicate that RAC3 variants result in morphological and functional defects in cortical neurons during brain development through variant-specific mechanisms, eventually leading to heterogeneous neurodevelopmental phenotypes.

Keywords: RAC3; axon guidance; brain development; neuronal migration; small GTPase.

  1. Department of Neurosciences, Rehabilitation, Ophthalmology, Genetics, Maternal and Child Health, University of Genoa, Genoa, Italy.
  2. Pediatric Neurology and Muscular Diseases Unit, IRCCS Istituto Giannina Gaslini, Genoa, Italy.
  3. Department of Molecular Neurobiology, Institute for Developmental Research, Aichi Developmental Disability Center, 713-8 Kamiya, Kasugai 480-0392, Japan.
  4. Genetics and Genome Biology, Research Institute, The Hospital for Sick Children, Toronto, Ontario, Canada.
  5. Department of Human Genetics, Emory Healthcare, Atlanta, GA 30322, USA.
  6. Genetics Laboratory, UDIAT-Centre Diagnòstic, Parc Taulí Hospital Universitari, Institut d’Investigació i Innovació Parc Taulí I3PT, Universitat Autònoma de, Barcelona, Sabadell, Spain.
  7. Dipartimento Universitario Scienze della Vita e Sanità Pubblica, Sezione di Medicina Genomica, Università Cattolica Sacro Cuore, Rome, Italy.
  8. Genetica Medica, Fondazione Policlinico Universitario A. Gemelli IRCCS, Rome, Italy.
  9. Neuropediatric Department, Helios-Klinikum Hildesheim, Hildesheim, Germany.
  10. Praxis für Humangenetik, Tübingen, Germany.
  11. GeneDx, Gaithersburg, MD, USA.
  12. Telethon Institute of Genetics and Medicine, Pozzuoli, Naples, Italy.
  13. Department of Precision Medicine, University of Campania Luigi Vanvitelli, Naples, Italy.
  14. INSERM UMR1231 Génétique des Anomalies du Développement, Université de Bourgogne Franche-Comté, Dijon, France.
  15. Laboratoire de Génétique Moléculaire, UF Innovation en diagnostic génomique des maladies rares, Plateau Technique de Biologie, CHU de Dijon Bourgogne, Dijon, France.
  16. Centre de Génétique et Centre de Référence Anomalies du Développement et Syndromes Malformatifs de l’interrégion Est, FHU TRANSLAD, Hôpital d’Enfants, CHU de Dijon Bourgogne, Dijon, France.
  17. Unit of Child Neuropsychiatry, Department of Medical and Surgical Neuroscience and Rehabilitation, IRCCS Istituto Giannina Gaslini, Genova, Italy.
  18. Division of Medical Genetics, Department of Pediatrics, Queen’s University, Kingston, ON K7L 2V7, Canada.
  19. Queen’s University, Kingston, ON, Canada.
  20. Paediatric Unit, Parc Taulí Hospital Universitari, Institut d’Investigació i Innovació Parc Taulí I3PT, Universitat Autònoma de, Barcelona, Sabadell, Spain.
  21. Department of Human Genetics, David Geffen School of Medicine, University of California-Los Angeles, Los Angeles, CA, USA.
  22. Department of Pediatrics, David Geffen School of Medicine, University of California-Los Angeles, Los Angeles, CA, USA.
  23. Department of Psychiatry and Biobehavioral Sciences, David Geffen School of Medicine, University of California-Los Angeles, Los Angeles, CA, USA.
  24. Department of Ophthalmology, Jules Stein Eye Institute, David Geffen School of Medicine, UCLA, Los Angeles 90095, CA, USA.
  25. Brain Research Institute, UCLA, Los Angeles 90095, CA, USA.
  26. Haseki Training and Research Hospital, Istanbul, Turkey.
  27. Division of Genomic Diagnostics, Department of Pathology and Laboratory Medicine, Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA.
  28. Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
  29. Pacific Northwest Research Institute, Seattle, WA 98122, USA.
  30. Baylor College of Medicine, Houston, TX 77030, USA.
  31. The Prenatal Diagnosis and Medical Genetics Program, Department of Obstetrics and Gynecology, Mount Sinai Hospital, University of Toronto, Toronto, Ontario, Canada.
  32. Division of Clinical and Metabolic Genetics, Department of Paediatrics, The Hospital for Sick Children, Toronto, Ontario, Canada.
  33. Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.
  34. Victorian Clinical Genetics Services, Murdoch Children’s Research Institute, and Department of Paediatrics, University of Melbourne, Melbourne, VIC 3052, Australia.
  35. Center for Medical Genetics, Ghent University Hospital, Gent, Belgium.
  36. Department of Pediatrics, University of California San Diego, San Diego, CA, USA.
  37. Genetics/Dysmorphology, Rady Children’s Hospital San Diego, San Diego, CA, USA.
  38. CeGaT GmbH, Tübingen, Germany.
  39. Neuroradiology Unit, IRCCS Istituto Giannina Gaslini, Genoa, Italy.
  40. Medical Genetics Unit, IRCCS Istituto Giannina Gaslini, Genoa, Italy.
  41. Department of Paediatrics, University of Toronto, Toronto, Ontario, Canada.
  42. Department of Neurochemistry, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Nagoya 466-8550, Japan.