通过分析SMN基因可变剪接与转录异构体表达探究脊肌萎缩症的分子致病机制

Spinal muscular atrophy (SMA) is the leading genetic cause of infant mortality, characterized by the degeneration of spinal motor neurons and muscle atrophy and with an incidence of 1/6000-1/10000.There is no effective therapy for SMA. Although the SMN2 gene is highly homologous to the pathogenic gene SMN1, the c.840C→T change on exon 7 leads the former to produce most of the transcripts with deletion of exon7 (SMN∆7) by abnormal alternative splicing. At present, it is unclear which relationship between the pattern of alternative splicing and kind of transcripts and the SMA phenotype. Our previous results showed that abnormal SMN transcribed isoforms presented in SMA patients compared to those in controls. In addition to the SMN∆7, we also found several novel transcript subtypes those had never been reported. At present, the role of alternative splicing and transcripts in the process of SMA is still unclear. In this project, the study cohorts will be 30-40 cases included normal controls, SMA carriers and patients. SMRT (single molecular real-time) sequencing technology was used to distinguish the extremely similar transcripts from the SMN1 and SMN2 gene. The complex splicing pattern and diverse transcripts disclosed here will be helpful for further understanding the molecular mechanism of SMA. The identification of new transcript subtypes may provide potential biomarkers for SMA. This study demonstrated a new option for identification of SMN1 and SMN2 transcripts. The information obtained in this study will be useful for the researchers conducted similar studies, such as identification of complicated genes with multi-copies or pseudogenes, and may provide potential targets for SMA gene therapy.

脊肌萎缩症(SMA)是临床婴儿致死性罕见遗传病，活婴发病率约1/10000，尚无有效疗法。其致病基因SMN1与修饰基因SMN2虽高度同源，但两者在编码区唯一差异c.840C→T却导致后者异常剪接并转录生成缺失7号外显子转录本SMN∆7。另有三篇文献提及单个碱基变异影响SMN1基因选择性剪接。除此之外，没有任何文献报道过SMN基因可变剪接和相应转录异构体与SMA疾病的分子关联。本课题组前期研究发现，SMA患者表达多种未见报道的新转录本亚型，表达谱显著区别于正常人。基于前期研究结果，本课题组拟收集正常人、携带者与患者样本30-40例，应用SMRT测序新技术，特异性分辨SMN1与SMN2基因高度相似转录本序列，鉴定新转录本亚型。从SMN基因可变剪接与转录异构体表达调控新视角出发，探究SMA疾病分子致病机制，为后续分子机理研究奠定重要基础，并为SMA基因治疗提供有用信息和潜在靶点。

脊肌萎缩症(SMA)是临床罕见婴儿致死性遗传病，活婴发病率约1/10000，目前尚无有效治疗方法。其致病基因SMN1与修饰基因SMN2虽高度同源，但两者在编码区唯一差异碱基c.840C→T却导致后者异常剪接并转录生成缺失7号外显子转录本SMN∆7。另有三篇文献提及单个碱基变异影响SMN1基因选择性剪接。除此之外，没有任何文献报道过SMN基因可变剪接和相应转录异构体与SMA疾病的分子关联。本课题联合应用多重连接探针扩增技术（MLPA）、定量PCR技术、Sanger测序、克隆测序、反转录PCR（RT-PCR）和单分子测序技术，完成了研究样本的SMN1基因拷贝数定量、SMN基因点突变鉴定、区别鉴定不同SMN基因来源的多种转录异构体并揭示其复杂多变的剪接模式。本课题首先构建了反转录等位基因特异性PCR（RT-AS-PCR）技术平台，用于SMN基因点突变的特异性鉴定，并为8例SMA家庭提供了明确的分子诊断，包括5例先证者和3例父母样本；其次，收集上述8例点突变家系，合并2例纯合缺失患者以及4例健康个体和1例SMN2点突变携带者，经克隆测序鉴定后，成功构建了单分子测序技术用于SMN基因转录异构体鉴定平台，该平台不仅能准确区分SMN1和SMN2来源的点突变，并且能有效鉴定高度同源的转录异构体。本课题最终鉴定出36种SMN基因转录异构体，其中SMN1和SMN2基因来源的各占一半。这些转录异构体中，18种为从未报道过的新型转录异构体。本研究还发现，无论是SMN1基因还是SMN2基因，都存在复杂的多重可变剪接模式，表现为单个或多个不连续外显子跳跃、内含子保留等模式。此外，SMN1和SMN2基因的转录表达谱也存在显著不同，前者主要转录全长SMN1（fill-length SMN）,而后者则主要表达不包含外显子7的转录本（SMNΔ7）。本课题构建的RT-AS-PCR平台可直接应用于临床SMA点突变病人的明确分子诊断；构建的单分子测序技术应用于SMN基因转录异构体鉴定平台可为后续相似的存在高度同源或假基因序列的基因转录异构体鉴定提供研究参考；本课题鉴定出的新型SMN基因转录异构体将为后续的SMA分子机理研究奠定重要基础，并为SMA基因治疗提供了潜在的分子标志物和研究靶点。