title: Characterisation of mutations of the phosphoinositide-3-kinase regulatory subunit, PIK3R2, in perisylvian polymicrogyria: a next-generation sequencing study.
abstract: BACKGROUND
  Bilateral perisylvian polymicrogyria (BPP), the most common form of regional polymicrogyria, causes the congenital bilateral perisylvian syndrome, featuring oromotor dysfunction, cognitive impairment, and epilepsy. The causes of BPP are heterogeneous, but only a few genetic causes have been reported. The aim of this study was to identify additional genetic causes of BPP and characterise their frequency in this population.
  
  
  METHODS
  Children (aged ≤18 years) with polymicrogyria were enrolled into our research programme from July, 1980, to October, 2015, at two centres (Florence, Italy, and Seattle, WA, USA). We obtained samples (blood and saliva) throughout this period at both centres and did whole-exome sequencing on DNA from eight trios (two parents and one affected child) with BPP in 2014. After the identification of mosaic PIK3R2 mutations in two of these eight children, we performed targeted screening of PIK3R2 by two methods in a cohort of 118 children with BPP. First, we performed targeted sequencing of the entire PIK3R2 gene by single molecule molecular inversion probes (smMIPs) on 38 patients with BPP with normal to large head size. Second, we did amplicon sequencing of the recurrent PIK3R2 mutation (Gly373Arg) in 80 children with various types of polymicrogyria including BPP. One additional patient had clinical whole-exome sequencing done independently, and was included in this study because of the phenotypic similarity to our cohort.
  
  
  RESULTS
  We identified a mosaic mutation (Gly373Arg) in a regulatory subunit of the PI3K-AKT-mTOR pathway, PIK3R2, in two children with BPP. Of the 38 patients with BPP and normal to large head size who underwent targeted next-generation sequencing by smMIPs, we identified constitutional and mosaic PIK3R2 mutations in 17 additional children. In parallel, one patient had the recurrent PIK3R2 mutation identified by clinical whole-exome sequencing. Seven of these 20 patients had BPP alone, and 13 had BPP in association with features of the megalencephaly-polymicrogyria-polydactyly-hydrocephalus (MPPH) syndrome. 19 patients had the same mutation (Gly373Arg), and one had a nearby missense mutation (Lys376Glu). Mutations were constitutional in 12 patients and mosaic in eight patients. In patients with mosaic mutations, we noted substantial variation in alternate (mutant) allele levels, ranging from ten (3%) of 377 reads to 39 (37%) of 106 reads, equivalent to 5-73% of cells analysed. Levels of mosaicism varied from undetectable to 37 (17%) of 216 reads in blood-derived DNA compared with 2030 (29%) of 6889 reads to 275 (43%) of 634 reads in saliva-derived DNA.
  
  
  CONCLUSIONS
  Constitutional and mosaic mutations in the PIK3R2 gene are associated with developmental brain disorders ranging from BPP with a normal head size to the MPPH syndrome. The phenotypic variability and low-level mosaicism, which challenge conventional molecular methods, have important implications for genetic testing and counselling.
  
  
  BACKGROUND
  US National Institutes of Health.
authors.affiliation: Division of Genetic Medicine, Department of Pediatrics, University of Washington, Seattle, WA, USA; Center for Integrative Brain Research, Seattle Children's Research Institute, Seattle, WA, USA. Electronic address: gmirzaa@uw.edu.
authors.forename: Ghayda M
authors.initials: GM
authors.lastname: Mirzaa
authors.affiliation: Pediatric Neurology, Neurogenetics and Neurobiology Unit and Laboratories, Neuroscience Department, A Meyer Children's Hospital, University of Florence, Florence, Italy.
authors.forename: Valerio
authors.initials: V
authors.lastname: Conti
authors.affiliation: Center for Developmental Biology and Regenerative Medicine, Seattle Children's Research Institute, Seattle, WA, USA.
authors.forename: Andrew E
authors.initials: AE
authors.lastname: Timms
authors.affiliation: Department of Neurology and Pediatrics, Washington University School of Medicine, St Louis, MO, USA.
authors.forename: Christopher D
authors.initials: CD
authors.lastname: Smyser
authors.affiliation: Center for Integrative Brain Research, Seattle Children's Research Institute, Seattle, WA, USA.
authors.forename: Sarah
authors.initials: S
authors.lastname: Ahmed
authors.affiliation: Division of Clinical and Metabolic Genetics, The Hospital for Sick Children, Toronto, ON, Canada.
authors.forename: Melissa
authors.initials: M
authors.lastname: Carter
authors.affiliation: Division of Medical Genetics, University of Missouri, St Louis, MO, USA.
authors.forename: Sarah
authors.initials: S
authors.lastname: Barnett
authors.affiliation: Division of Human Genetics, Cincinnati Children's Hospital, Cincinnati, OH, USA.
authors.forename: Robert B
authors.initials: RB
authors.lastname: Hufnagel
authors.affiliation: Division of Child Neurology, Children's Hospital of Pittsburgh, Pittsburgh, PA, USA.
authors.forename: Amy
authors.initials: A
authors.lastname: Goldstein
authors.affiliation: Department of Pediatrics, Shimada Ryoiku Center Hachioji, Tokyo, Japan.
authors.forename: Yoko
authors.initials: Y
authors.lastname: Narumi-Kishimoto
authors.affiliation: Center for Integrative Brain Research, Seattle Children's Research Institute, Seattle, WA, USA.
authors.forename: Carissa
authors.initials: C
authors.lastname: Olds
authors.affiliation: Center for Integrative Brain Research, Seattle Children's Research Institute, Seattle, WA, USA.
authors.forename: Sarah
authors.initials: S
authors.lastname: Collins
authors.affiliation: Genetics Department, Permanente Medical Group, San Francisco, CA, USA.
authors.forename: Kathreen
authors.initials: K
authors.lastname: Johnston
authors.affiliation: Centre National de Génotypage, Evry, France.
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Deleuze
authors.affiliation: Plateforme de Bioinformatique Paris-Descartes, Institut Imagine, Paris, France.
authors.forename: Patrick
authors.initials: P
authors.lastname: Nitschké
authors.affiliation: Genetics and Molecular Pathology, Women's and Children's Hospital, North Adelaide, SA, Australia.
authors.forename: Kathryn
authors.initials: K
authors.lastname: Friend
authors.affiliation: Division of Medical Genetics, University of Missouri, St Louis, MO, USA.
authors.forename: Catharine
authors.initials: C
authors.lastname: Harris
authors.affiliation: Division of Genetics, Birth Defects and Metabolism, Ann and Robert H Lurie Children's Hospital of Chicago, Chicago, IL, USA.
authors.forename: Allison
authors.initials: A
authors.lastname: Goetsch
authors.affiliation: Department of Genome Sciences, University of Washington, Seattle, WA, USA.
authors.forename: Beth
authors.initials: B
authors.lastname: Martin
authors.affiliation: Department of Genetics, Stanford University School of Medicine, Stanford, CA, USA.
authors.forename: Evan August
authors.initials: EA
authors.lastname: Boyle
authors.affiliation: Pediatric Neurology, Neurogenetics and Neurobiology Unit and Laboratories, Neuroscience Department, A Meyer Children's Hospital, University of Florence, Florence, Italy.
authors.forename: Elena
authors.initials: E
authors.lastname: Parrini
authors.affiliation: Pediatric Neurology, Neurogenetics and Neurobiology Unit and Laboratories, Neuroscience Department, A Meyer Children's Hospital, University of Florence, Florence, Italy.
authors.forename: Davide
authors.initials: D
authors.lastname: Mei
authors.affiliation: Pediatric Neurology, Neurogenetics and Neurobiology Unit and Laboratories, Neuroscience Department, A Meyer Children's Hospital, University of Florence, Florence, Italy.
authors.forename: Lorenzo
authors.initials: L
authors.lastname: Tattini
authors.affiliation: Department of Pediatrics, Division of Genetics, University of California, San Francisco, CA, USA.
authors.forename: Anne
authors.initials: A
authors.lastname: Slavotinek
authors.affiliation: Department of Clinical Genetics, Churchill Hospital, Oxford University Hospitals, Headington, UK.
authors.forename: Ed
authors.initials: E
authors.lastname: Blair
authors.affiliation: South Australian Clinical Genetics Service, Women's and Children's Hospital/SA Pathology, North Adelaide, SA, Australia; Discipline of Pediatrics, University of Adelaide, Adelaide, Australia.
authors.forename: Christopher
authors.initials: C
authors.lastname: Barnett
authors.affiliation: Department of Genome Sciences, University of Washington, Seattle, WA, USA.
authors.forename: Jay
authors.initials: J
authors.lastname: Shendure
authors.affiliation: Pôle de biologie, Hôpitaux Universitaires de Strasbourg, Strasbourg, France; IGBMC, Translational Medicine and Neurogenetics Department, Illkirch, France.
authors.forename: Jamel
authors.initials: J
authors.lastname: Chelly
authors.affiliation: Division of Genetic Medicine, Department of Pediatrics, University of Washington, Seattle, WA, USA; Center for Integrative Brain Research, Seattle Children's Research Institute, Seattle, WA, USA.
authors.forename: William B
authors.initials: WB
authors.lastname: Dobyns
authors.affiliation: Pediatric Neurology, Neurogenetics and Neurobiology Unit and Laboratories, Neuroscience Department, A Meyer Children's Hospital, University of Florence, Florence, Italy; IRCCS Stella Maris Foundation, Pisa, Italy. Electronic address: renzo.guerrini@meyer.it.
authors.forename: Renzo
authors.initials: R
authors.lastname: Guerrini
mesh_terms: D000015:Abnormalities, Multiple / Q000235:genetics*; D000293:Adolescent; D002648:Child; D002675:Child, Preschool; D004252:DNA Mutational Analysis; D006801:Humans; D007223:Infant; D008607:Intellectual Disability / Q000235:genetics*; D054220:Malformations of Cortical Development / Q000235:genetics*; D009154:Mutation; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics*; D055815:Young Adult
chemical_list: D019869:Phosphatidylinositol 3-Kinases; C560566:phosphoinositol-3 kinase regulatory subunit 2, human