Association of MTOR Mutations With Developmental Brain Disorders, Including Megalencephaly, Focal Cortical Dysplasia, and Pigmentary Mosaicism.
Association of MTOR Mutations With Developmental Brain Disorders, Including Megalencephaly, Focal Cortical Dysplasia, and Pigmentary Mosaicism.
复制标题
DOI：
10.1001/jamaneurol.2016.0363
复制
发表时间：
2016-07-01
期刊：
影响因子：
29
通讯作者：
Dobyns WB
中科院分区：
文献类型：
--
作者：
Mirzaa GM;Campbell CD;Solovieff N;Goold C;Jansen LA;Menon S;Timms AE;Conti V;Biag JD;Adams C;Boyle EA;Collins S;Ishak G;Poliachik S;Girisha KM;Yeung KS;Chung BHY;Rahikkala E;Gunter SA;McDaniel SS;Macmurdo CF;Bernstein JA;Martin B;Leary R;Mahan S;Liu S;Weaver M;Doerschner M;Jhangiani S;Muzny DM;Boerwinkle E;Gibbs RA;Lupski JR;Shendure J;Saneto RP;Novotny EJ;Wilson CJ;Sellers WR;Morrissey M;Hevner RF;Ojemann JG;Guerrini R;Murphy LO;Winckler W;Dobyns WB
局灶性皮质发育不良(FCD)、半巨脑(HMEG)和巨脑是一系列皮质发育畸形，具有共同的神经病理特征。总体而言，这些疾病与儿童发病率和死亡率显著相关。尤其是FCD，是儿童难治性局灶性癫痫最常见的原因。确定FCD、HMEG和弥漫性巨脑的潜在分子病因学。我们对8例FCD或HMEG患儿进行了全外显子组测序(WES)，测序采用标准深度(~50-60X)测序，从患儿及其父母的外周血样(血液、唾液或皮肤)和脑组织进行深度(~150-180X)测序。我们使用靶向测序和WES筛查了93名患有分子原因不明的弥漫性或局灶性脑过度生长的儿童(42名FCD-HMEG，51名弥漫性巨脑)。对切除的脑组织和培养的神经元进行PI3K-AKT-MTOR通路活性的组织病理学和功能分析，以验证突变。整个外显子组测序和靶向测序确定了与这一发育脑部疾病谱相关的变异。我们在四名患有2a型功能性精神障碍的儿童脑组织中发现了低水平的MTOR型马赛克突变，可选的等位基因比例从0.012到0.086不等。我们还在三名无血缘关系的弥漫性巨脑和皮肤色素性马赛克样伊藤黑素病的儿童中发现了MTOR的中等水平马赛克突变(p.Thr1977Ile)。最后，我们在三名患有弥漫性巨脑和智力残疾的无关儿童中发现了MTOR的一种新生突变(p.Glu1799Lys)。对两名2a型FCD型儿童的分子和功能分析显示，在最易致痫的区域有一个震中的交替等位基因片段的梯度。当在培养的神经元中表达时，这里发现的所有MTOR突变都会驱动mTORC1的结构性激活和神经元体积增大，从而在患者中发现MTOR突变和神经元肥大之间的联系。MTORC1抑制剂RAD001可改善这些表型。我们的数据显示，MTOR突变与从FCD2型到弥漫性巨脑畸形的一系列脑过度生长表型相关，通过不同的突变和嵌合体水平来区分。这些突变足以导致培养神经元的细胞肥大。我们的数据还提供了一个令人信服的大脑马赛克模式的证明，并证实了MTOR的马赛克突变与皮肤中的色素马赛克之间的联系。
Focal cortical dysplasia (FCD), hemimegalencephaly (HMEG) and megalencephaly constitute a spectrum of malformations of cortical development with shared neuropathologic features. Collectively, these disorders are associated with significant childhood morbidity and mortality. FCD, in particular, represents the most frequent cause of intractable focal epilepsy in children. To identify the underlying molecular etiology of FCD, HMEG, and diffuse megalencephaly. We performed whole exome sequencing (WES) on eight children with FCD or HMEG using standard depth (~50-60X) sequencing in peripheral samples (blood, saliva or skin) from the affected child and their parents, and deep (~150-180X) sequencing in affected brain tissue. We used both targeted sequencing and WES to screen a cohort of 93 children with molecularly unexplained diffuse or focal brain overgrowth (42 with FCD-HMEG, and 51 with diffuse megalencephaly). Histopathological and functional assays of PI3K-AKT-MTOR pathway activity in resected brain tissue and cultured neurons were performed to validate mutations. Whole exome sequencing and targeted sequencing identified variants associated with this spectrum of developmental brain disorders. We identified low-level mosaic mutations of MTOR in brain tissue in four children with FCD type 2a with alternative allele fractions ranging from 0.012–0.086. We also identified intermediate level mosaic mutation of MTOR (p.Thr1977Ile) in three unrelated children with diffuse megalencephaly and pigmentary mosaicism in skin that resembles hypomelanosis of Ito. Finally, we identified a constitutional de novo mutation of MTOR (p.Glu1799Lys) in three unrelated children with diffuse megalencephaly and intellectual disability. Molecular and functional analysis in two children with FCD type 2a from whom multiple affected brain tissue samples were available revealed a gradient of alternate allele fractions with an epicenter in the most epileptogenic area. When expressed in cultured neurons, all MTOR mutations identified here drive constitutive activation of mTORC1 and enlarged neuronal size, establishing a link between the MTOR mutations and neuronal hypertrophy found in patients. The mTORC1 inhibitor RAD001 ameliorated these phenotypes. Our data show that mutations of MTOR are associated with a spectrum of brain overgrowth phenotypes extending from FCD type 2 to diffuse megalencephaly, distinguished by different mutations and levels of mosaicism. These mutations are sufficient to cause cellular hypertrophy in cultured neurons. Our data also provide a compelling demonstration of the pattern of mosaicism in brain, and substantiate the link between mosaic mutations of MTOR and pigmentary mosaicism in skin.