Conti Valerio, Carabalona Aurelie, Pallesi-Pocachard Emilie, Leventer Richard J, Schaller Fabienne, Parrini Elena, Deparis Agathe A, Watrin Françoise, Buhler Emmanuelle, Novara Francesca, Lise Stefano, Pagnamenta Alistair T, Kini Usha, Taylor Jenny C, Zuffardi Orsetta, Represa Alfonso, Keays David Antony, Guerrini Renzo, Falace Antonio, Cardoso Carlos
University of Florence.
INSERM INMED; Aix-Marseille University; Columbia University.
J Vis Exp. 2017 Dec 1(130):53570. doi: 10.3791/53570.
Birth defects that involve the cerebral cortex - also known as malformations of cortical development (MCD) - are important causes of intellectual disability and account for 20-40% of drug-resistant epilepsy in childhood. High-resolution brain imaging has facilitated in vivo identification of a large group of MCD phenotypes. Despite the advances in brain imaging, genomic analysis and generation of animal models, a straightforward workflow to systematically prioritize candidate genes and to test functional effects of putative mutations is missing. To overcome this problem, an experimental strategy enabling the identification of novel causative genes for MCD was developed and validated. This strategy is based on identifying candidate genomic regions or genes via array-CGH or whole-exome sequencing and characterizing the effects of their inactivation or of overexpression of specific mutations in developing rodent brains via in utero electroporation. This approach led to the identification of the C6orf70 gene, encoding for a putative vesicular protein, to the pathogenesis of periventricular nodular heterotopia, a MCD caused by defective neuronal migration.
涉及大脑皮层的出生缺陷——也称为皮质发育畸形(MCD)——是智力残疾的重要原因,占儿童耐药性癫痫的20%-40%。高分辨率脑成像有助于在体内识别一大类MCD表型。尽管在脑成像、基因组分析和动物模型生成方面取得了进展,但仍缺乏一个直接的工作流程来系统地对候选基因进行优先级排序并测试假定突变的功能效应。为了克服这个问题,开发并验证了一种能够识别MCD新致病基因的实验策略。该策略基于通过阵列比较基因组杂交(array-CGH)或全外显子测序识别候选基因组区域或基因,并通过子宫内电穿孔表征其失活或特定突变在发育中的啮齿动物大脑中过表达的影响。这种方法导致了编码假定囊泡蛋白的C6orf70基因与室管膜下结节性异位症的发病机制相关,室管膜下结节性异位症是一种由神经元迁移缺陷引起的MCD。