Robust quantification of the SMN gene copy number by real-time TaqMan PCR
Robust quantification of the SMN gene copy number by real-time TaqMan PCR
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DOI：
10.1007/s10048-007-0093-1
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发表时间：
2007-11-01
期刊：
影响因子：
2.2
通讯作者：
Wang, Wenlan
中科院分区：
文献类型：
--
作者：
Gomez-Curet, Ilsa;Robinson, Karyn G.;Wang, Wenlan
脊髓性肌萎缩症（SMA）是由存活运动神经元基因1 （SMN1）突变或缺失引起的常染色体隐性遗传病。高度同源的基因SMN2存在于所有患者中，但它不能弥补SMN1的缺失。SMN2与SMN1的不同之处在于几个核苷酸的变化，但外显子7的C -> T转换导致外显子跳变。因此，大多数SMN2基因转录本缺少第7外显子。虽然SMN1是疾病的决定基因，SMN2拷贝的数量似乎调节SMA的临床表型。因此，确定SMN拷贝数对临床诊断和预后具有重要意义。我们开发了一种用于SMN1和SMN2基因的实时定量TaqMan聚合酶链反应方法，可以对两种不同分离方法和两种不同来源（人血液和皮肤成纤维细胞）获得的脱氧核糖核酸样品进行可靠的拷贝数测定。对于SMN1，等位基因特异性仅通过添加等位基因特异性前向引物来实现，对于SMN2，通过添加特异性前向引物和非延伸寡核苷酸（SMN1阻滞剂）来将SMN1的非特异性扩增降低到可忽略的水平。我们验证了这种实时聚合酶链反应方法的可靠性，并发现所有基因拷贝数测量的变异系数都低于10%。通过这种方法对SMA成纤维细胞中SMN拷贝数的定量分析显示SMN1的缺失和SMN2拷贝数与疾病严重程度呈负相关。
Spinal muscular atrophy (SMA) is an autosomal recessive disease caused by mutation or deletion of the survival motor neuron gene 1 (SMN1). The highly homologous gene, SMN2, is present in all patients, but it cannot compensate for loss of SMN1. SMN2 differs from SMN1 by a few nucleotide changes, but a C -> T transition in exon 7 leads to exon skipping. As a result, most transcripts from the SMN2 gene lack exon 7. Although SMN1 is the disease-determining gene, the number of SMN2 copies appears to modulate SMA clinical phenotypes. Thus, determining the SMN copy number is important for clinical diagnosis and prognosis. We have developed a quantitative real-time TaqMan polymerase chain reaction assay for both the SMN1 and SMN2 genes, in which reliable copy number determination was possible on deoxyribonucleic acid samples obtained by two different isolation methods and from two different sources (human blood and skin fibroblasts). For SMN1, allele specificity was attained solely by addition of an allele-specific forward primer and, for SMN2, by addition of a specific forward primer and a nonextending oligonucleotide (SMN1 blocker) that reduced nonspecific amplification from SMN1 to a negligible level. We validated the reliability of this real-time polymerase chain reaction approach and found that the coefficient of variation for all the gene copy number measurements was below 10%. Quantitative analysis of the SMN copy number in SMA fibroblasts by this approach showed deletion of SMN1 and an inverse correlation between the SMN2 copy number and severity of the disease.