New multiplex real-time PCR approach to detect gene mutations for spinal muscular atrophy.
New multiplex real-time PCR approach to detect gene mutations for spinal muscular atrophy.
复制标题
新的多重实时 PCR 方法检测脊髓性肌萎缩症基因突变
DOI：
10.1186/s12883-016-0651-y
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发表时间：
2016-08-17
期刊：
影响因子：
2.6
通讯作者：
Zou L
中科院分区：
文献类型：
--
作者：
Liu Z;Zhang P;He X;Liu S;Tang S;Zhang R;Wang X;Tan J;Peng B;Jiang L;Hong S;Zou L
脊髓性肌萎缩症（SMA）是儿童最常见的常染色体隐性遗传病，诊断复杂、困难，尤其是早期。早期诊断SMA能够改善SMA患者的预后。本研究采用实时荧光定量PCR技术检测SMA关键基因的突变或缺失，并进一步分析基因型-表型相关性。方法建立检测运动神经元存活（SMN）、凋亡抑制蛋白（NAIP）和通用转录因子IIH、多肽2基因（GTF2H2）突变的多重实时荧光定量PCR方法，并通过DNA测序和多重连接依赖探针扩增（MLPA）进行验证。本方法对141例住院儿童、100例正常儿童及2000例干血斑（DBS）标本的诊断及预后进行了分析。结果建立的多重实时荧光定量PCR检测smn、naipandgtf2h2基因突变的准确率与DNA测序和MLPA相比至少为98.8%。141例肢体运动障碍患儿中，75例为SMA。71例SMA伴smnc，占94.67%。840例突变，9例（12%）为naipdeletion， 3例（4%）为gtf2h2deletion。多重实时荧光定量PCR能够诊断和预测SMA患者的预后。同时，应用实时荧光定量PCR检测DBS的微量DNA，能够对SMA进行早期诊断。结论总结了西南地区SMA的临床及分子特征。我们的工作为实时PCR检测儿童SMA提供了一种新的方法，并有望用于新生儿SMA早期诊断的筛查。
BackgroundSpinal muscular atrophy (SMA) is the most common autosomal recessive disease in children, and the diagnosis is complicated and difficult, especially at early stage. Early diagnosis of SMA is able to improve the outcome of SMA patients. In our study, Real-time PCR was developed to measure the gene mutation or deletion of key genes for SMA and to further analyse genotype-phenotype correlation.MethodsThe multiple real-time PCR for detecting the mutations of survival of motor neuron (SMN), apoptosis inhibitory protein (NAIP) and general transcription factor IIH, polypeptide 2 gene (GTF2H2) was established and confirmed by DNA sequencing and multiplex ligation-dependent probe amplification (MLPA). The diagnosis and prognosis of 141 hospitalized children, 100 normal children and further 2000 cases of dry blood spot (DBS) samples were analysed by this multiple real-time PCR.ResultsThe multiple real-time PCR was established and the accuracy of it to detect the mutations ofSMN,NAIPandGTF2H2was at least 98.8 % comparing with DNA sequencing and MLPA. Among 141 limb movement disorders children, 75 cases were SMA. 71 cases of SMA (94.67 %) were withSMNc.840 mutation, 9 cases (12 %) withNAIPdeletion and 3 cases (4 %) withGTF2H2deletion. The multiple real-time PCR was able to diagnose and predict the prognosis of SMA patients. Simultaneously, the real-time PCR was applied to detect trace DNA from DBS and able to make an early diagnosis of SMA.ConclusionThe clinical and molecular characteristics of SMA in Southwest of China were presented. Our work provides a novel way for detecting SMA in children by using real-time PCR and the potential usage in newborn screening for early diagnosis of SMA.