Objective To perform mutation analysis of survival motor neuron gene 1 (SMN1 in two spinal muscular atrophy (SMA) patients and their parents to evaluate the effects of the two SMN1 gene mutations on the transcript levels of the gene and preliminarily predict their effects on the structure and function of SMN protein.Methods Mutation analysis of SMN1 gene was carried out by multiplex ligationdependent probe amplification,reverse transcript-polymerase chain reaction (RT-PCR) and cloning sequencing.Transmission of the mutations was confirmed by the mutation analysis in patients' parents.The full-length SMN1 (SMN1-fl) transcript levels of the patients carrying these subtle mutations were detected using quantitative RT-PCR.Results The two patients were diagnosed as SMA Ⅱ and SMA Ⅲ.They carried p.Val19GlyfsX21 and p.Ala2Gly SMN1 mutations in SMN1 gene,respectively.Both of the two mutations were originated from their fathers.Compared with the healthy individuals (23.5 ± 4.9),the two patients had a significant reduction in the level of SMN1-fl transcripts (t =3.322,P =0.011 (p.Ala2Gly) ;t =6.964,P =0.000 (p.Val19GlyfsX21)).However,compared with the healthy carriers (14.1 ±4.5),the patient with p.Ala2Gly mutation had no significant reduction in the level of SMN1-fl transcripts (13.9 ±3.6,t =0.058,P =0.955) ; however,the patient with p.Val19GlyfsX21 mutation had a significant reduction (4.9± 2.4,t =3.725,P =0.004).Conclusions Two SMN1 gene mutations are identified in our study.The mutation p.Val19GlyfsX21 is a novel mutation and p.Ala2Gly is firstly reported in Chinese SMA patients.p.Val19GlyfsX21 may possibly lead to decreased SMN1-fl mRNA by nonsense-mediated messenger RNA decay,however,p.Ala2Gly has no obvious effects on the amount of the SMN1-fl transcripts,indicating that its deleterious effect may be occurring at SMN protein level or the function of SMN protein.%目的 对2例脊髓性肌萎缩症(SMA)患者及其核心家系进行运动神经元存活基因1(SMN1)的点突变分析,评估2种SMN1点突变对全长SMN1基因(SMN1-fl)转录水平的影响,结合患者表型初步预测点突变对SMN蛋白及功能的影响.方法 采用多重连接依赖性探针扩增技术、RTPCR、克隆测序结合基因组序列分析进行SMN1基因拷贝数和点突变分析,利用患者父母的基因分析明确突变的传递关系.通过实时定量PCR技术分析患者体内SMN1-fl的转录水平.以50名无神经肌肉萎缩病史的个体作为健康对照组,健康个体为含2个SMN1基因拷贝的个体,而健康携带者为纯合缺失患儿的父母(含1个SMN1基因拷贝).结果 2例SMA患者的表型为Ⅱ型和Ⅲ型,SMN1基因分别存在p.Val19GlyfsX21和p.Ala2Gly突变,突变均来自患者父亲.健康个体、健康携带者、携带p.Val19GlyfsX21突变的患者和携带p.Ala2Gly突变的患者,其SMN1-fl mRNA表达量分别为23.5±4.9、14.1±4.5、4.9±2.4和13.9±3.6.t检验结果表明,携带上述突变的2例患者,其SMN1-flmRNA表达量均明显低于健康个体(t=3.322,P=0.011;t =6.964,P=0.000),而与健康携带者相比,p.Ala2Gly突变的患者SMN1-fl mRNA表达量差异无统计学意义(t=0.058,P=0.955),携带p.Val19GlyfsX21突变的患者其SMN1-fl mRNA表达量显著下降,差异有统计学意义(t=3.725,P=0.004).结论 共发现2种SMN1基因突变,p.Val19GlyfsX21为国际尚未见报道的新突变,p.Ala2Gly突变首次在中国患者中发现.前者可能通过无义突变介导的mRNA降解途径使SMN1-fl mRNA转录水平显著下降,而后者则并不影响SMN1-fl mRNA转录水平,对SMN蛋白功能的影响尚待研究.
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