Institutes of Biomedical Science, Shanxi University, Taiyuan, China.
Mol Genet Genomic Med. 2019 Sep;7(9):e858. doi: 10.1002/mgg3.858. Epub 2019 Jul 22.
Beta-propeller protein-associated neurodegeneration (BPAN, OMIM 300894) is an X-linked neurodegenerative disorder caused by mutations in WDR45. WDR45 is required for autophagy, defect in WDR45 impaired autophagy which contributes for the pathogenesis of BPAN. Previously, we reported a novel de novo mutation (c.1040_1041del, p.Glu347GlyfsTer7) in WDR45 (NM_007075) in a 3-year-old Chinese girl with BPAN.
The protein structure was constructed using SWISS-MODEL and the isoelectric point (pI) was predicted by the online pI/Mw tool at ExPASy. The functional effects of this mutation were predicted by two online software programs: PROVEN and MutationTaster. Stable overexpression of Flag-tagged wild-type or mutant WDR45 in HeLa cells was constructed. Protein levels of LC3 and p62 were analyzed by western blot upon treatment with/without autophagy inhibitor Bafilomycin A1, the formation of LC3 puncta were analyzed in HeLa cells transfected with mCherry-LC3 by confocal microscopy.
The mutation resulted in a shift of pI from 6.74 to 8.84 and was predicted to be pathogenic. The protein levels of LC3-II and p62 were increased in cells overexpression of wild-type and mutant WDR45 while the protein levels were not increased in cells overexpression of mutant WDR45 upon treatment with autophagy inhibitor Bafilomycin A1. Results from confocal microscopy revealed that LC3-positive puncta were increased in cells expressing both wild-type and mutant WDR45 while the number of LC3-positive puncta was not increased in cells expressing mutant WDR45 upon treatment with Bafilomycin A1.
Our study evidenced that this novel mutation in WDR45 impaired autophagy in cells thus this mutation is the cause for BPAN in this patient.
β-三叶螺旋蛋白相关神经退行性疾病（BPAN，OMIM 300894）是一种 X 连锁神经退行性疾病，由 WDR45 基因突变引起。WDR45 是自噬所必需的，WDR45 的缺陷会损害自噬，这有助于 BPAN 的发病机制。此前，我们报道了一名 3 岁中国女孩 BPAN 中 WDR45（NM_007075）的一个新的从头突变（c.1040_1041del，p.Glu347GlyfsTer7）。
使用 SWISS-MODEL 构建蛋白质结构，使用在线 pI/Mw 工具预测等电点（pI）。使用两个在线软件程序：PROVEN 和 MutationTaster 预测该突变的功能影响。在 HeLa 细胞中构建 Flag 标记的野生型或突变型 WDR45 的稳定过表达。用自噬抑制剂 Bafilomycin A1 处理或不处理后，通过 Western blot 分析 LC3 和 p62 的蛋白水平，通过共聚焦显微镜分析 HeLa 细胞中转染 mCherry-LC3 后 LC3 点状的形成。
该突变导致 pI 从 6.74 移位到 8.84，预测为致病性。在过表达野生型和突变型 WDR45 的细胞中，LC3-II 和 p62 的蛋白水平增加，而在用自噬抑制剂 Bafilomycin A1 处理后，过表达突变型 WDR45 的细胞中 LC3-II 和 p62 的蛋白水平没有增加。共聚焦显微镜的结果表明，在表达野生型和突变型 WDR45 的细胞中，LC3 阳性点状增多，而在用 Bafilomycin A1 处理后，表达突变型 WDR45 的细胞中 LC3 阳性点状增多没有增加。
本研究表明，WDR45 中的这个新突变会损害细胞中的自噬，因此该突变是该患者 BPAN 的原因。
