Department of Neurology and Institute of Neurology, Rui Jin Hospital & Rui Jin Hospital North, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
Department of Neurology and Institute of Neurology, Rui Jin Hospital & Rui Jin Hospital North, Shanghai Jiao Tong University School of Medicine, Shanghai, China; Department of Neurology, Suzhou Hospital Affiliated to Anhui Medical University, Suzhou, China.
Parkinsonism Relat Disord. 2019 Dec;69:125-133. doi: 10.1016/j.parkreldis.2019.11.007. Epub 2019 Nov 6.
Mutations in the SPAST gene are the most frequent cause of hereditary spastic paraplegia (HSP). We aim to extend the mutation spectrum of spastic paraplegia 4 (SPG4) and carried out experiment in vitro to explore the influence of the SPAST gene mutation on the function of corresponding protein.
Whole-exome sequencing (WES) combined with multiplex ligation-dependent probe amplification (MLPA) were performed in a cohort of 150 patients clinically diagnosed with HSP. We focus on screening for mutations in SPAST gene and carrying out functional experiments to assess the effects of the novel variants.
A total of 34 different mutations in the SPAST gene were identified, of which 10 were novel, including 1 missense (c.1479T > A), 1 nonsense (c.766G > T), 3 splicing (c.1413 + 1_1413+4delGTAA, c.1729-1G > A and c.1536+2T > G) and 5 frameshift mutations (c.1094delC, c.885dupA, c.517_518delAG, c.280delG and c.908dupC). For 7 novel non-splicing mutations, functional study showed that accumulated M1 spastin colcocalized with microtubules which was different from a uniformly diffused M87 spastin. While an impairment in severing activity was observed in both mutant M1 and mutant M87, except for c.280delG. All 3 novel splicing variants w ere predicted to affect splicing by using bioinformatic programs. However, only c.1536+2T > G had no influence on splice site in vitro, which conflicts with the in-silico analysis.
We genetically diagnosed 40 SPG4 patients. All the novel non-splicing mutations except for c.280delG were certified to exert an effect on the microtubule-severing and all the novel splicing mutations other than c.1536+2T > G would cause abnormal splicing of the spastin.
SPAST 基因突变是遗传性痉挛性截瘫（HSP）最常见的原因。我们旨在扩展痉挛性截瘫 4 型（SPG4）的突变谱，并进行体外实验以探究 SPAST 基因突变对相应蛋白功能的影响。
对 150 例临床诊断为 HSP 的患者进行全外显子组测序（WES）结合多重连接依赖性探针扩增（MLPA）。我们专注于筛查 SPAST 基因突变，并进行功能实验以评估新变异体的影响。
共鉴定出 SPAST 基因中的 34 种不同突变，其中 10 种是新的，包括 1 种错义突变（c.1479T>G），1 种无义突变（c.766G>T），3 种剪接突变（c.1413+1_1413+4delGTAA、c.1729-1G>A 和 c.1536+2T>G）和 5 种移码突变（c.1094delC、c.885dupA、c.517_518delAG、c.280delG 和 c.908dupC）。对于 7 种新的非剪接突变，功能研究表明，积累的 M1 痉挛蛋白与微管共定位，与均匀扩散的 M87 痉挛蛋白不同。尽管在突变体 M1 和突变体 M87 中都观察到切割活性受损，但除了 c.280delG 以外。所有 3 种新的剪接变体都被预测会通过生物信息学程序影响剪接。然而，只有 c.1536+2T>G 在体外对剪接位点没有影响，这与计算机分析结果相矛盾。
我们对 40 例 SPG4 患者进行了基因诊断。除 c.280delG 外，所有新的非剪接突变均被证明对微管切割有影响，除 c.1536+2T>G 外，所有新的剪接突变都会导致痉挛蛋白的异常剪接。
