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  3. A combination of genetic and biochemical analyses for the diagnosis of PI3K-AKT-mTOR pathway-associated megalencephaly

A combination of genetic and biochemical analyses for the diagnosis of PI3K-AKT-mTOR pathway-associated megalencephaly

  • BMC Med Genet. 2017 Jan 13;18(1):4. doi: 10.1186/s12881-016-0363-6.
Yutaka Negishi 1,  Fuyuki Miya 2 3,  Ayako Hattori 1,  Yoshikazu Johmura 4 5,  Motoo Nakagawa 6,  Naoki Ando 1,  Ikumi Hori 1,  Takao Togawa 1,  Kohei Aoyama 1,  Kei Ohashi 1,  Shinobu Fukumura 7,  Seiji Mizuno 8,  Ayako Umemura 9,  Yoko Kishimoto 10,  Nobuhiko Okamoto 11,  Mitsuhiro Kato 12 13,  Tatsuhiko Tsunoda 2 3,  Mami Yamasaki 14,  Yonehiro Kanemura 15 16,  Kenjiro Kosaki 17,  Makoto Nakanishi 3 5,  Shinji Saitoh 18
Affiliations

Affiliations

  • 1 Department of Pediatrics and Neonatology, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuho-Cho, Mizuho-Ku, Nagoya, 467-8601, Japan.
  • 2 Department of Medical Science Mathematics, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan.
  • 3 Laboratory for Medical Science Mathematics, RIKEN Center for Integrative Medical Sciences, Yokohama, Japan.
  • 4 Department of Cell Biology, Graduate School of Medical Sciences, Nagoya City University, Nagoya, Japan.
  • 5 Present address: Division of Cancer Cell Biology, Department of Cancer Biology, Instuite of Medical Science, The University of Tokyo, Tokyo, Japan.
  • 6 Department of Radiology, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan.
  • 7 Department of Pediatrics, Sapporo Medical University School of Medicine, Sapporo, Japan.
  • 8 Department of Pediatrics, Central Hospital, Aichi Human Service Center, Aichi, Japan.
  • 9 Department of Pediatric Neurology, Central Hospital, Aichi Human Service Center, Aichi, Japan.
  • 10 Department of Pediatrics, Shimada Ryoiku Center Hachiouji, Tokyo, Japan.
  • 11 Department of Medical Genetics, Osaka Medical Center and Research Institute for Maternal and Child Health, Osaka, Japan.
  • 12 Department of Pediatrics, Yamagata University Faculty of Medicine, Yamagata, Japan.
  • 13 Present address: Department of Pediatrics, Showa University School of Medicine, Tokyo, Japan.
  • 14 Department of Neurosurgery, Takatsuki General Hospital, Osaka, Japan.
  • 15 Division of Regenerative Medicine, Institute for Clinical Research, Osaka National Hospital, National Hospital Organization, Osaka, Japan.
  • 16 Department of Neurosurgery, Osaka National Hospital, National Hospital Organization, Osaka, Japan.
  • 17 Center for Medical Genetics, Keio University School of Medicine, Tokyo, Japan.
  • 18 Department of Pediatrics and Neonatology, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuho-Cho, Mizuho-Ku, Nagoya, 467-8601, Japan. [email protected].
PMID: 28086757 DOI: 10.1186/s12881-016-0363-6
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.

Keywords

AKT3; MCAP; MPPH; Multiplex targeted sequencing; PIK3R2; PTEN; Phosphorylated S6 ribosomal protein.

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