title: A combination of genetic and biochemical analyses for the diagnosis of PI3K-AKT-mTOR pathway-associated megalencephaly.
abstract: BACKGROUND
  Constitutive activation of the PI3K-AKT-mTOR pathway (mTOR pathway) underlies megalencephaly in many patients. Yet, prevalence of the involvement of the PI3K-AKT-mTOR pathway in patients with megalencephaly remains to be elucidated, and molecular diagnosis is challenging. Here, we have successfully established a combination of genetic and biochemical methods for diagnosis of mTOR pathway-associated megalencephaly, and have attempted to delineate the clinical characteristics of the disorder.
  
  
  METHODS
  Thirteen patients with an increased head circumference and neurological symptoms participated in the study. To evaluate the activation of the mTOR pathway, we performed western blot analysis to determine the expression levels of phosphorylated S6 ribosomal protein (phospho-S6 protein) in lymphoblastoid cell lines from 12 patients. Multiplex targeted sequencing analysis for 15 genes involved in the mTOR pathway was performed on 12 patients, and whole-exome sequencing was performed on one additional patient. Clinical features and MRI findings were also investigated.
  
  
  RESULTS
  We identified pathogenic mutations in six (AKT3, 1 patient; PIK3R2, 2 patients; PTEN, 3 patients) of the 13 patients. Increased expression of phospho-S6 protein was demonstrated in all five mutation-positive patients in whom western blotting was performed, as well as in three mutation-negative patients. Developmental delay, dysmorphic facial features were observed in almost all patients. Syndactyly/polydactyly and capillary malformations were not observed, even in patients with AKT3 or PIK3R2 mutations. There were no common phenotypes or MRI findings among these patients.
  
  
  CONCLUSIONS
  A combination of genetic and biochemical methods successfully identified mTOR pathway involvement in nine of 13 (approximately 70%) patients with megalencephaly, indicating a major contribution of the pathway to the pathogenesis of megalencephaly. Our combined approach could be useful to identify patients who are suitable for future clinical trials using an mTOR inhibitor.
authors.affiliation: Department of Pediatrics and Neonatology, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuho-Cho, Mizuho-Ku, Nagoya, 467-8601, Japan.
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Negishi
authors.affiliation: Department of Medical Science Mathematics, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan.|Laboratory for Medical Science Mathematics, RIKEN Center for Integrative Medical Sciences, Yokohama, Japan.
authors.forename: Fuyuki
authors.initials: F
authors.lastname: Miya
authors.affiliation: Department of Pediatrics and Neonatology, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuho-Cho, Mizuho-Ku, Nagoya, 467-8601, Japan.
authors.forename: Ayako
authors.initials: A
authors.lastname: Hattori
authors.affiliation: Department of Cell Biology, Graduate School of Medical Sciences, Nagoya City University, Nagoya, Japan.|Present address: Division of Cancer Cell Biology, Department of Cancer Biology, Instuite of Medical Science, The University of Tokyo, Tokyo, Japan.
authors.forename: Yoshikazu
authors.initials: Y
authors.lastname: Johmura
authors.affiliation: Department of Radiology, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan.
authors.forename: Motoo
authors.initials: M
authors.lastname: Nakagawa
authors.affiliation: Department of Pediatrics and Neonatology, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuho-Cho, Mizuho-Ku, Nagoya, 467-8601, Japan.
authors.forename: Naoki
authors.initials: N
authors.lastname: Ando
authors.affiliation: Department of Pediatrics and Neonatology, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuho-Cho, Mizuho-Ku, Nagoya, 467-8601, Japan.
authors.forename: Ikumi
authors.initials: I
authors.lastname: Hori
authors.affiliation: Department of Pediatrics and Neonatology, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuho-Cho, Mizuho-Ku, Nagoya, 467-8601, Japan.
authors.forename: Takao
authors.initials: T
authors.lastname: Togawa
authors.affiliation: Department of Pediatrics and Neonatology, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuho-Cho, Mizuho-Ku, Nagoya, 467-8601, Japan.
authors.forename: Kohei
authors.initials: K
authors.lastname: Aoyama
authors.affiliation: Department of Pediatrics and Neonatology, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuho-Cho, Mizuho-Ku, Nagoya, 467-8601, Japan.
authors.forename: Kei
authors.initials: K
authors.lastname: Ohashi
authors.affiliation: Department of Pediatrics, Sapporo Medical University School of Medicine, Sapporo, Japan.
authors.forename: Shinobu
authors.initials: S
authors.lastname: Fukumura
authors.affiliation: Department of Pediatrics, Central Hospital, Aichi Human Service Center, Aichi, Japan.
authors.forename: Seiji
authors.initials: S
authors.lastname: Mizuno
authors.affiliation: Department of Pediatric Neurology, Central Hospital, Aichi Human Service Center, Aichi, Japan.
authors.forename: Ayako
authors.initials: A
authors.lastname: Umemura
authors.affiliation: Department of Pediatrics, Shimada Ryoiku Center Hachiouji, Tokyo, Japan.
authors.forename: Yoko
authors.initials: Y
authors.lastname: Kishimoto
authors.affiliation: Department of Medical Genetics, Osaka Medical Center and Research Institute for Maternal and Child Health, Osaka, Japan.
authors.forename: Nobuhiko
authors.initials: N
authors.lastname: Okamoto
authors.affiliation: Department of Pediatrics, Yamagata University Faculty of Medicine, Yamagata, Japan.|Present address: Department of Pediatrics, Showa University School of Medicine, Tokyo, Japan.
authors.forename: Mitsuhiro
authors.initials: M
authors.lastname: Kato
authors.affiliation: Department of Medical Science Mathematics, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan.|Laboratory for Medical Science Mathematics, RIKEN Center for Integrative Medical Sciences, Yokohama, Japan.
authors.forename: Tatsuhiko
authors.initials: T
authors.lastname: Tsunoda
authors.affiliation: Department of Neurosurgery, Takatsuki General Hospital, Osaka, Japan.
authors.forename: Mami
authors.initials: M
authors.lastname: Yamasaki
authors.affiliation: Division of Regenerative Medicine, Institute for Clinical Research, Osaka National Hospital, National Hospital Organization, Osaka, Japan.|Department of Neurosurgery, Osaka National Hospital, National Hospital Organization, Osaka, Japan.
authors.forename: Yonehiro
authors.initials: Y
authors.lastname: Kanemura
authors.affiliation: Center for Medical Genetics, Keio University School of Medicine, Tokyo, Japan.
authors.forename: Kenjiro
authors.initials: K
authors.lastname: Kosaki
authors.affiliation: Laboratory for Medical Science Mathematics, RIKEN Center for Integrative Medical Sciences, Yokohama, Japan.|Present address: Division of Cancer Cell Biology, Department of Cancer Biology, Instuite of Medical Science, The University of Tokyo, Tokyo, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Nakanishi
authors.affiliation: Department of Pediatrics and Neonatology, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuho-Cho, Mizuho-Ku, Nagoya, 467-8601, Japan. ss11@med.nagoya-cu.ac.jp.
authors.forename: Shinji
authors.initials: S
authors.lastname: Saitoh
mesh_terms: D000293:Adolescent; D002460:Cell Line; D002648:Child; D002675:Child, Preschool; D005260:Female; D006801:Humans; D008297:Male; D058627:Megalencephaly / Q000175:diagnosis* / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D051059:PTEN Phosphohydrolase / Q000235:genetics* / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics*; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics* / Q000378:metabolism; D019893:Ribosomal Protein S6 Kinases / Q000378:metabolism*; D017422:Sequence Analysis, DNA / Q000379:methods; D015398:Signal Transduction; D058570:TOR Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism
chemical_list: D051059:PTEN Phosphohydrolase; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D019893:Ribosomal Protein S6 Kinases; D058570:TOR Serine-Threonine Kinases; C560566:phosphoinositol-3 kinase regulatory subunit 2, human; C546842:MTOR protein, human; C491754:AKT3 protein, human; C494929:PTEN protein, human
keywords: AKT3; MCAP; MPPH; Multiplex targeted sequencing; PIK3R2; PTEN; Phosphorylated S6 ribosomal protein