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.
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DOI：
10.1186/s12881-016-0363-6
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发表时间：
2017-01-13
影响因子：
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通讯作者：
Saitoh S
中科院分区：
文献类型：
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作者：
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
PI 3 K-AKT-mTOR通路（mTOR通路）的组成性激活是许多患者中巨脑畸形的基础。然而，巨脑畸形患者中PI 3 K-AKT-mTOR通路参与的患病率仍有待阐明，分子诊断具有挑战性。在这里，我们已经成功地建立了一个基因和生化方法相结合的mTOR通路相关的巨脑畸形的诊断，并试图描绘疾病的临床特征。13名头围增加和神经系统症状的患者参加了这项研究。为了评估mTOR通路的激活，我们进行了蛋白质印迹分析，以确定来自12名患者的淋巴母细胞样细胞系中磷酸化S6核糖体蛋白（磷酸-S6蛋白）的表达水平。对12名患者进行了涉及mTOR通路的15个基因的多重靶向测序分析，并对另外1名患者进行了全外显子组测序。并对患者的临床表现及MRI表现进行分析。我们在13例患者中的6例（AKT 3，1例患者; PIK 3R 2，2例患者; PTEN，3例患者）中确定了致病性突变。磷酸-S6蛋白表达增加，证明在所有5个突变阳性的患者进行蛋白质印迹，以及在3个突变阴性的患者。几乎所有患者均出现发育迟缓、面部畸形。即使在AKT 3或PIK 3R 2突变患者中，也未观察到并指/多指和毛细血管畸形。这些患者没有共同的表型或MRI表现。遗传学和生物化学方法的组合成功地确定了mTOR通路参与9/13例（约70%）巨脑畸形患者，表明该通路对巨脑畸形发病机制的主要贡献。我们的综合方法可能有助于确定适合未来使用mTOR抑制剂进行临床试验的患者。
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. 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. 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. 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.