The SMN1 common variant c.22 dupA in Chinese patients causes spinal muscular atrophy by nonsense-mediated mRNA decay in humans
The SMN1 common variant c.22 dupA in Chinese patients causes spinal muscular atrophy by nonsense-mediated mRNA decay in humans
复制标题
中国患者中的 SMN1 常见变异 c.22 dupA 通过无义介导的 mRNA 衰变导致人类脊髓性肌萎缩
DOI：
10.1016/j.gene.2017.10.048
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发表时间：
2018-02-20
期刊：
影响因子：
3.5
通讯作者：
Song, Fang
中科院分区：
文献类型：
--
作者：
Bai, JinLi;Qu, YuJin;Song, Fang
脊髓性肌萎缩症（SMA）是一种常见的常染色体隐性神经肌肉疾病，主要由SMN1基因纯合缺失引起。大约5%-10%的SMA患者被认为有SMN1变异。c.22 dupA （p.Ser8lysfs*23）已被确定为中国SMA人群中最常见的变异，并与严重的表型相关。然而，该变异在SMA发病中的确切分子机制尚不清楚。我们观察到c.22 dupA患者外周血中SMN1 mRNA和SMN蛋白含量低于对照组。本研究的目的是探讨无义介导的mRNA衰变（NMD）是否在SMN1基因c.22 dupA变异引起SMA的机制中起作用。来自患者1和患者2的两个c.22 dupA患者的两个淋巴母细胞系和来自患者2的一个真皮成纤维细胞系纳入我们的研究。提供了NMD机制的两阶段验证。我们首先用NMD通路抑制剂（包括嘌呤霉素和环血红素）治疗SMA患者的永生化b淋巴母细胞和真皮成纤维细胞后，通过实时定量PCR测量SMN1基因转录水平的变化。接下来，在成纤维细胞系中进行慢病毒介导的NMD关键因子-上移码蛋白1 （UPF1）的敲低，以进一步阐明该变体是否导致NMD，因为UPF1在翻译过程中将异常终止的转录物识别为NMD底物。SC35 1.7 kb转录本，一个生理NMD底物在我们的实验中被确定为NMD阳性基因。这两种抑制剂导致全长SMN1 （ft-SMN1）转录本水平的急剧上升。此外，SC35 1.7-kb mRNA水平也增加，表明NMD通路受到两种抑制剂的抑制。对于3种细胞系，两种淋巴母细胞和一种成纤维细胞，环己亚胺SMN1转录物水平分别增加2.5 +/- 0.4 ~ 8.3 +/- 0.1、1.9 +/- 0.2 ~ 5.0 +/- 0.7和2.2 +/- 0.1 ~ 4.9 +/- 0.2倍。在这些细胞系中，purromycin SMN1转录物水平的增加倍数分别为5.5 +/- 0.2 ~ 19.5 +/- 4.0、3.1 +/- 0.3 ~ 9.9 +/- 1.8和1.5 +/- 0.2 ~ 6.5 +/- 0.5。同时，SC35 1.7-kb转录水平在3种细胞系中均显著升高。此外，与阴性对照慢病毒相比，慢病毒介导的UPF1敲低导致UPF1蛋白水平降低22.5%。此外，UPF1基因的敲低也促进了SC35 1.7kb和fl-SMN1基因的mRNA表达。在患者2的成纤维细胞中，SMN1和SC35 1.7-kb mRNA水平分别增加了约4倍和6.5倍。总之，我们的研究提供了SMN1基因c.22 dupA变异触发NMD的第一个证据。患者的SMA发病机制与SMN1的mRNA降解有关，而与截断的SMN蛋白无关。
Spinal muscular atrophy (SMA) is a common autosomal recessive neuromuscular disorder that is mostly caused by homozygous deletion of the SMN1 gene. Approximately 5%-10% of SMA patients are believed to have SMN1 variants. c.22 dupA (p.Ser8lysfs*23) has been identified as the most frequent variant in the Chinese SMA population and to be associated with a severe phenotype. However, the exact molecular mechanism of the variant on the pathogenesis of SMA is unclear. We observed that SMN1 mRNA and the SMN protein in the peripheral blood cells of a patient with c.22 dupA were lower than those of controls. The aim of this study is to investigate whether nonsense-mediated mRNA decay (NMD) plays a role in the mechanism of the c.22 dupA variant of the SMN1 gene as it causes SMA. Two lymphoblasts cell lines from two patients (patient 1 and 2) with the c.22 dupA, and one dermal fibroblasts cell line from patient 2 were included in our study. Two-stage validation of the NMD mechanism was supplied. We first measured the changes in the transcript levels of the SMN1 gene by real-time quantitative PCR after immortalized B-lymphoblasts and dermal fibroblasts cells of the SMA patients were treated with inhibitors of the NMD pathway, including puromycin and cyclohemide. Next, lentivirus-mediated knockdown of the key NMD factor-Up-frameshift protein 1 (UPF1)-was performed in the fibroblasts cell line to further clarify whether the variant led to NMD, as UPF1 recognizes abnormally terminated transcripts as NMD substrates during translation. SC35 1.7-kb transcripts, a physiological NMD substrate was determined to be a NMD positive gene in our experiments. The two inhibitors resulted in a dramatic escalation of the levels of the full-length SMN1 (ft-SMN1) transcripts. Additionally, the SC35 1.7-kb mRNA levels were also increased, suggesting that NMD pathway is suppressed by the two inhibitors. For the 3 cell lines, the fold increase of the SMN1 transcript levels of cycloheximide ranged from 2.5 +/- 0.4 to 8.3 +/- 0.1, 1.9 +/- 0.2 to 5.0 +/- 0.7 and 2.2 +/- 0.1 to 4.9 +/- 0.2 for two lymphoblastoid cell lines and one fibroblasts cell line, respectively. For these cell lines, the fold increases of the SMN1 transcript levels of puromycin were as follows: 5.5 +/- 0.2 to 19.5 +/- 4.0, 3.1 +/- 0.3 to 9.9 +/- 1.8 and 1.5 +/- 0.2 to 6.5 +/- 0.5. Meanwhile, the SC35 1.7-kb transcript levels were markedly increased in all 3 cell lines. In addition, lentivirus-mediated UPF1 knockdown lead to a reduction of the UPF1 protein level to 22.5% compared to the negative control lentivirus. Additionally, knockdown of the UPF1 gene also promoted mRNA expression of the SC35 1.7kb and fl-SMN1 genes. The increases of the SMN1 and SC35 1.7-kb mRNA levels reached about 4- and 6.5-fold in fibroblasts derived from the patient 2, respectively. Altogether, our study provides the first evidence that the c.22 dupA variant in the SMN1 gene triggers NMD. SMA pathogenesis in the patient is associated with mRNA degradation of SMN1, but not the truncated SMN protein.