BACKGROUND 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. METHODS 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. RESULTS 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. CONCLUSION 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.

中文翻译：

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SPAST基因中的新突变会导致遗传性痉挛性截瘫。

背景技术SPAST基因的突变是遗传性痉挛性截瘫（HSP）的最常见原因。我们旨在扩大痉挛性截瘫4（SPG4）的突变谱，并进行体外实验，以探索SPAST基因突变对相应蛋白功能的影响。方法在临床诊断为HSP的150例患者中，进行了全外显子测序（WES）与多重连接依赖探针扩增（MLPA）的结合。我们专注于筛选SPAST基因中的突变，并进行功能实验以评估新变体的影响。结果在SPAST基因中总共鉴定出34个不同的突变，其中10个是新颖的，包括1个错义（c.1479T> A），1个无意义（c.766G> T），3个剪接（c.1413 + 1_1413 + 4delGTAA，c.1729-1G> A和c。1536 + 2T> G）和5个移码突变（c.1094delC，c.885dupA，c.517_518delAG，c.280delG和c.908dupC）。对于7个新的非剪接突变，功能研究表明，累积的M1 spastin与微管共聚集，这与均匀扩散的M87 spastin不同。尽管在突变体M1和突变体M87中都观察到了切断活性的损伤，但c.280delG除外。通过使用生物信息学程序，可以预测所有3种新颖的剪接变体都会影响剪接。但是，只有c.1536 + 2T> G对体外剪接位点没有影响，这与计算机内分析相冲突。结论我们通过遗传学诊断了40例SPG4患者。除c.280delG外，所有新的非剪接突变均被证明对微管切割有作用，除c.1536 + 2T以外的所有新的剪接突变均>