Lim Jae Seok, Gopalappa Ramu, Kim Se Hoon, Ramakrishna Suresh, Lee Minji, Kim Woo-Il, Kim Junho, Park Sang Min, Lee Junehawk, Oh Jung-Hwa, Kim Heung Dong, Park Chang-Hwan, Lee Joon Soo, Kim Sangwoo, Kim Dong Seok, Han Jung Min, Kang Hoon-Chul, Kim Hyongbum Henry, Lee Jeong Ho
Brain Korea 21 Plus Project, Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science & Technology, Daejeon 34141, South Korea.
Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul 04763, South Korea; Brain Korea 21 Plus Project for Medical Sciences, Graduate Program of Nano Science and Technology, Department of Pharmacology, Yonsei University College of Medicine, Seoul 03722, South Korea.
Am J Hum Genet. 2017 Mar 2;100(3):454-472. doi: 10.1016/j.ajhg.2017.01.030. Epub 2017 Feb 16.
Focal cortical dysplasia (FCD) is a major cause of the sporadic form of intractable focal epilepsies that require surgical treatment. It has recently been reported that brain somatic mutations in MTOR account for 15%-25% of FCD type II (FCDII), characterized by cortical dyslamination and dysmorphic neurons. However, the genetic etiologies of FCDII-affected individuals who lack the MTOR mutation remain unclear. Here, we performed deep hybrid capture and amplicon sequencing (read depth of 100×-20,012×) of five important mTOR pathway genes-PIK3CA, PIK3R2, AKT3, TSC1, and TSC2-by using paired brain and saliva samples from 40 FCDII individuals negative for MTOR mutations. We found that 5 of 40 individuals (12.5%) had brain somatic mutations in TSC1 (c.64C>T [p.Arg22Trp] and c.610C>T [p.Arg204Cys]) and TSC2 (c.4639G>A [p.Val1547Ile]), and these results were reproducible on two different sequencing platforms. All identified mutations induced hyperactivation of the mTOR pathway by disrupting the formation or function of the TSC1-TSC2 complex. Furthermore, in utero CRISPR-Cas9-mediated genome editing of Tsc1 or Tsc2 induced the development of spontaneous behavioral seizures, as well as cytomegalic neurons and cortical dyslamination. These results show that brain somatic mutations in TSC1 and TSC2 cause FCD and that in utero application of the CRISPR-Cas9 system is useful for generating neurodevelopmental disease models of somatic mutations in the brain.
局灶性皮质发育不良（FCD）是需要手术治疗的散发性难治性局灶性癫痫的主要病因。最近有报道称，MTOR基因的脑体细胞突变占II型FCD（FCDII）的15%-25%，其特征为皮质板层结构紊乱和神经元异形。然而，缺乏MTOR突变的FCDII患者的遗传病因仍不清楚。在此，我们使用来自40名MTOR突变阴性的FCDII患者的配对脑和唾液样本，对五个重要的mTOR通路基因——PIK3CA、PIK3R2、AKT3、TSC1和TSC2进行了深度杂交捕获和扩增子测序（读取深度为100×-20,012×）。我们发现，40名患者中有5名（12.5%）在TSC1（c.64C>T [p.Arg22Trp]和c.610C>T [p.Arg204Cys]）和TSC2（c.4639G>A [p.Val1547Ile]）存在脑体细胞突变，这些结果在两个不同的测序平台上均可重现。所有鉴定出的突变均通过破坏TSC1-TSC2复合物的形成或功能诱导mTOR通路的过度激活。此外，子宫内CRISPR-Cas9介导的Tsc1或Tsc2基因编辑可诱导自发行为性癫痫发作的发生，以及巨细胞神经元和皮质板层结构紊乱。这些结果表明，TSC1和TSC2的脑体细胞突变可导致FCD，并且子宫内应用CRISPR-Cas9系统有助于建立脑体细胞突变的神经发育疾病模型。
