Accuracy of Marker Analysis, Quantitative Real-Time Polymerase Chain Reaction, and Multiple Ligation-Dependent Probe Amplification to Determine SMN2 Copy Number in Patients with Spinal Muscular Atrophy
Accuracy of Marker Analysis, Quantitative Real-Time Polymerase Chain Reaction, and Multiple Ligation-Dependent Probe Amplification to Determine SMN2 Copy Number in Patients with Spinal Muscular Atrophy
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DOI：
10.1089/gtmb.2010.0253
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发表时间：
2011-09-01
影响因子：
1.4
通讯作者：
Tizzano, Eduardo F.
中科院分区：
文献类型：
--
作者：
Alias, Laura;Bernal, Sara;Tizzano, Eduardo F.
脊髓性肌萎缩症（SMA）是一种常染色体隐性遗传性神经肌肉疾病，由运动神经元存活基因（SMN 1）缺失或突变引起。所有SMA患者均具有高度同源的SMN 1（SMN 2基因）拷贝。重度（I型）SMA患者存在1个或2个SMN 2拷贝，而轻度慢性型（II-III型）患者通常存在3个或4个SMN 2拷贝。SMN 2剂量对于运动功能检查和临床试验的患者分层很重要。我们的目的是比较三种方法，标记物分析，实时定量聚合酶链反应，使用LightCycler仪器，和多重连接依赖性探针扩增（MLPA），以表征其在定量SMN 2基因的准确性。我们研究了一组62例经遗传学证实的SMA患者，其中54例SMN 1外显子7和8纯合缺失，8例SMN 2-SMN 1杂合基因缺失。在32例患者（51.6%）中观察到使用三种方法的完全相关性。在其余30例患者中，发现三种方法之间存在不一致性，包括标记物分析相对于定量方法（LightCycler和MLPA）低估或高估SMN 2拷贝，因为缺乏标记物的信息性、SMN基因的3'缺失和SMN 2-SMN 1杂合基因中的断点。讨论了这些方法的技术局限性和优缺点。我们得出结论，这三种方法在大多数情况下相互补充，估计SMN 2拷贝数。然而，MLPA提供了额外的信息来表征SMA病例的特殊重排，如部分缺失和杂交基因。
Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder caused by absence of or mutations in the survival motor neuron1 gene (SMN1). All SMA patients have a highly homologous copy of SMN1, the SMN2 gene. Severe (type I) SMA patients present one or two SMN2 copies, whereas milder chronic forms (type II-III) usually have three or four SMN2 copies. SMN2 dosage is important to stratify patients for motor function tests and clinical trials. Our aim was to compare three methods, marker analysis, real-time quantitative polymerase chain reaction using the LightCycler instrument, and multiple ligation-dependent probe amplification (MLPA), to characterize their accuracy in quantifying SMN2 genes. We studied a group of 62 genetically confirmed SMA patients, 54 with homozygous absence of exons 7 and 8 of SMN1 and 8 with SMN2-SMN1 hybrid genes. A complete correlation using the three methods was observed in 32 patients (51.6%). In the remaining 30 patients, discordances between the three methods were found, including under or overestimation of SMN2 copies by marker analysis with respect to the quantitative methods (LightCycler and MLPA) because of lack of informativeness of markers, 3' deletions of SMN genes, and breakpoints in SMN2-SMN1 hybrid genes. The technical limitations and advantages and disadvantages of these methods are discussed. We conclude that the three methods complement each other in estimating the SMN2 copy number in most cases. However, MLPA offers additional information to characterize SMA cases with particular rearrangements such as partial deletions and hybrid genes.