Determination of SMN1 and SMN2 copy number using TaqMan™ technology
Determination of SMN1 and SMN2 copy number using TaqMan™ technology
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DOI：
10.1002/humu.10221
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发表时间：
2003-07-01
期刊：
影响因子：
3.9
通讯作者：
Zerres, K
中科院分区：
文献类型：
--
作者：
Anhuf, D;Eggermann, T;Zerres, K
婴儿脊髓性肌萎缩症（SMA）是一种神经肌肉疾病，90%以上的患者因 SMN1 基因纯合缺失而引起。鉴定 SMN1 缺失携带者对于诊断目的和遗传咨询非常重要。目前 SMN2 拷贝数测定的实际意义有限，但可能很重要，例如对于未来的药物试验。在这里，我们提出了一种新的快速可靠的方法来确定 SMN1 和 SMN2 基因的拷贝数：为了区分这两个基因，我们使用小沟结合剂 (MGB) 探针在 TaqManT1 技术的基础上开发了一种定量测试。为了评估检测 SMN1 缺失携带者的方法，我们测试了 40 名假定携带者以及 100 名对照。我们确认了所有个人的携带者状态；此外，对照组中 SMN1 和 SMN2 拷贝的分布与之前发布的一致。总的来说，两个基因的不同拷贝数范围之间存在明显的差异。这种明显的区别是基于 MGB 探针的精确特异性和外部参考基因座的平行分析，避免了 DNA 浓度不可避免的偏差问题。这里介绍的 TaqMan 测定的简单性和可重复性应有助于其在分子诊断实验室中的建立。然而，基于 SMN 区域的复杂性和 SMA 的临床变异性，SMN 拷贝数定量分析的适用性需要了解其选择和局限性。因此，SMN1 和 SMN2 拷贝数的确定只能在仔细考虑每种情况后才提供。 (C) 2003 Wiley-Liss, Inc.
Infantile spinal muscular atrophy (SMA) is a neuromuscular disease caused by homozygous deletion of the SMN1 gene in more than 90% of patients. Identification of carriers for the SMN1 deletion is important for diagnostic purposes and for genetic counseling. The current practical implications of SMN2 copy number determination are limited but may be important, for example, for future drug trials. Here we present a new rapid and reliable approach to determine the copy numbers of the SMN1 and SMN2 genes: For differentiation of the two genes, we developed a quantitative test on the basis of TaqManT1 technology using minor groove binder (MGB) probes. To evaluate the approach in respect to detection of SMN1 deletion carriers, we tested 40 putative carriers as well as 100 controls. We confirmed the carrier status in all individuals; furthermore, the distribution of SMN1 and SMN2 copies in the control cohort corresponded to that published previously. In total, a clear,cut differentiation between the different copy number ranges could be observed for both genes. This distinct differentiation is based on the exact specificity of MGB probes and the parallel analysis of an external reference locus that circumvents the problem of unavoidable deviations in DNA concentrations. The simplicity and reproducibility of the TaqMan assay presented here should facilitate its establishment in molecular diagnostic laboratories. Nevertheless, the applicability of quantitative analyses of SMN copy numbers requires knowledge about its options and limitations, based on the complex nature of the SMN region and the clinical variability of SMA. Therefore, determination of SMN1 and SMN2 copy numbers should only be offered after careful consideration in each case. (C) 2003 Wiley-Liss, Inc.