Yokoi Setsuri, Ishihara Naoko, Miya Fuyuki, Tsutsumi Makiko, Yanagihara Itaru, Fujita Naoko, Yamamoto Hiroyuki, Kato Mitsuhiro, Okamoto Nobuhiko, Tsunoda Tatsuhiko, Yamasaki Mami, Kanemura Yonehiro, Kosaki Kenjiro, Kojima Seiji, Saitoh Shinji, Kurahashi Hiroki, Natsume Jun
Division of Molecular Genetics, Institute for Comprehensive Medical Science, Fujita Health University, Toyoake, Japan.
Department of Pediatrics, Nagoya University Graduate School of Medicine, Nagoya, Japan.
Sci Rep. 2015 Oct 23;5:15165. doi: 10.1038/srep15165.
TUBA1A mutations cause a wide spectrum of lissencephaly and brain malformations. Here, we report two patients with severe cortical dysgeneses, one with an extremely thin cerebral parenchyma apparently looking like hydranencephaly and the other with lissencephaly accompanied by marked hydrocephalus, both harbouring novel de novo missense mutations of TUBA1A. To elucidate how the various TUBA1A mutations affect the severity of the phenotype, we examined the capacity of the mutant protein to incorporate into the endogenous microtubule network in transfected COS7 cells by measuring line density using line extraction in an immunofluorescence study. The mutants responsible for severe phenotypes were found to incorporate extensively into the network. To determine how each mutant alters the microtubule stability, we examined cold-induced microtubule depolymerisation in fibroblasts. The depolymerisation of patients' fibroblasts occurred earlier than that of control fibroblasts, suggesting that microtubules bearing mutated tubulins are unstable. Both mutations are predicted to participate in lateral interactions of microtubules. Our data suggest that the TUBA1A mutations disrupting lateral interactions have pronounced dominant-negative effects on microtubule dynamics that are associated with the severe end of the lissencephaly spectrum.
TUBA1A突变会导致多种类型的无脑回畸形和脑畸形。在此,我们报告了两名患有严重皮质发育异常的患者,一名患者的脑实质极薄,外观类似积水性无脑畸形,另一名患者患有无脑回畸形并伴有明显脑积水,两人均携带新的TUBA1A新生错义突变。为了阐明各种TUBA1A突变如何影响表型的严重程度,我们通过在免疫荧光研究中使用线条提取法测量线条密度,检测了突变蛋白在转染的COS7细胞中整合到内源性微管网络中的能力。发现导致严重表型的突变体广泛整合到网络中。为了确定每个突变体如何改变微管稳定性,我们检测了成纤维细胞中冷诱导的微管解聚情况。患者成纤维细胞的解聚比对照成纤维细胞更早发生,这表明携带突变微管蛋白的微管不稳定。两种突变预计都参与微管的横向相互作用。我们的数据表明,破坏横向相互作用的TUBA1A突变对微管动力学具有明显的显性负性作用,这与无脑回畸形谱系的严重端相关。