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

Yutaka Negishi, Fuyuki Miya, Ayako Hattori, Yoshikazu Johmura, Motoo Nakagawa, Naoki Ando, Ikumi Hori, Takao Togawa, Kohei Aoyama, Kei Ohashi, Shinobu Fukumura, Seiji Mizuno, Ayako Umemura, Yoko Kishimoto, Nobuhiko Okamoto, Mitsuhiro Kato, Tatsuhiko Tsunoda, Mami Yamasaki, Yonehiro Kanemura, Kenjiro KosakiMakoto Nakanishi, Shinji Saitoh

Research output: Contribution to journalArticle

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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.

Original languageEnglish
Article number4
JournalBMC Medical Genetics
Volume18
Issue number1
DOIs
Publication statusPublished - 2017 Jan 13

Fingerprint

Megalencephaly
Phosphatidylinositol 3-Kinases
Molecular Biology
S 6
Mutation
Western Blotting
Ribosomal Protein S6
Syndactyly
Exome
Polydactyly

Keywords

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

ASJC Scopus subject areas

  • Genetics
  • Genetics(clinical)

Cite this

A combination of genetic and biochemical analyses for the diagnosis of PI3K-AKT-mTOR pathway-associated megalencephaly. / Negishi, Yutaka; Miya, Fuyuki; Hattori, Ayako; Johmura, Yoshikazu; Nakagawa, Motoo; Ando, Naoki; Hori, Ikumi; Togawa, Takao; Aoyama, Kohei; Ohashi, Kei; Fukumura, Shinobu; Mizuno, Seiji; Umemura, Ayako; Kishimoto, Yoko; Okamoto, Nobuhiko; Kato, Mitsuhiro; Tsunoda, Tatsuhiko; Yamasaki, Mami; Kanemura, Yonehiro; Kosaki, Kenjiro; Nakanishi, Makoto; Saitoh, Shinji.

In: BMC Medical Genetics, Vol. 18, No. 1, 4, 13.01.2017.

Research output: Contribution to journalArticle

Negishi, Y, Miya, F, Hattori, A, Johmura, Y, Nakagawa, M, Ando, N, Hori, I, Togawa, T, Aoyama, K, Ohashi, K, Fukumura, S, Mizuno, S, Umemura, A, Kishimoto, Y, Okamoto, N, Kato, M, Tsunoda, T, Yamasaki, M, Kanemura, Y, Kosaki, K, Nakanishi, M & Saitoh, S 2017, 'A combination of genetic and biochemical analyses for the diagnosis of PI3K-AKT-mTOR pathway-associated megalencephaly', BMC Medical Genetics, vol. 18, no. 1, 4. https://doi.org/10.1186/s12881-016-0363-6
Negishi, Yutaka ; Miya, Fuyuki ; Hattori, Ayako ; Johmura, Yoshikazu ; Nakagawa, Motoo ; Ando, Naoki ; Hori, Ikumi ; Togawa, Takao ; Aoyama, Kohei ; Ohashi, Kei ; Fukumura, Shinobu ; Mizuno, Seiji ; Umemura, Ayako ; Kishimoto, Yoko ; Okamoto, Nobuhiko ; Kato, Mitsuhiro ; Tsunoda, Tatsuhiko ; Yamasaki, Mami ; Kanemura, Yonehiro ; Kosaki, Kenjiro ; Nakanishi, Makoto ; Saitoh, Shinji. / A combination of genetic and biochemical analyses for the diagnosis of PI3K-AKT-mTOR pathway-associated megalencephaly. In: BMC Medical Genetics. 2017 ; Vol. 18, No. 1.
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T1 - A combination of genetic and biochemical analyses for the diagnosis of PI3K-AKT-mTOR pathway-associated megalencephaly

AU - Negishi, Yutaka

AU - Miya, Fuyuki

AU - Hattori, Ayako

AU - Johmura, Yoshikazu

AU - Nakagawa, Motoo

AU - Ando, Naoki

AU - Hori, Ikumi

AU - Togawa, Takao

AU - Aoyama, Kohei

AU - Ohashi, Kei

AU - Fukumura, Shinobu

AU - Mizuno, Seiji

AU - Umemura, Ayako

AU - Kishimoto, Yoko

AU - Okamoto, Nobuhiko

AU - Kato, Mitsuhiro

AU - Tsunoda, Tatsuhiko

AU - Yamasaki, Mami

AU - Kanemura, Yonehiro

AU - Kosaki, Kenjiro

AU - Nakanishi, Makoto

AU - Saitoh, Shinji

PY - 2017/1/13

Y1 - 2017/1/13

N2 - 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.

AB - 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.

KW - AKT3

KW - MCAP

KW - MPPH

KW - Multiplex targeted sequencing

KW - Phosphorylated S6 ribosomal protein

KW - PIK3R2

KW - PTEN

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