Anhuf Dirk, Eggermann Thomas, Rudnik-Schöneborn Sabine, Zerres Klaus
Institute of Human Genetics, RWTH Aachen, Aachen, Germany.
Hum Mutat. 2003 Jul;22(1):74-8. doi: 10.1002/humu.10221.
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 TaqMan 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.
婴儿脊髓性肌萎缩症（SMA）是一种神经肌肉疾病，90%以上的患者由SMN1基因纯合缺失所致。鉴定SMN1基因缺失的携带者对于诊断及遗传咨询具有重要意义。目前，SMN2基因拷贝数测定的实际应用有限，但可能具有重要意义，例如对未来的药物试验。在此，我们提出一种快速可靠的新方法来测定SMN1和SMN2基因的拷贝数：为区分这两个基因，我们基于TaqMan技术，使用小沟结合剂（MGB）探针开发了一种定量检测方法。为评估该方法在检测SMN1基因缺失携带者方面的效果，我们检测了40名疑似携带者以及100名对照者。我们确认了所有个体的携带者状态；此外，对照队列中SMN1和SMN2基因拷贝数的分布与先前发表的结果一致。总体而言，两个基因在不同拷贝数范围之间均可观察到明显区分。这种明显区分基于MGB探针的精确特异性以及对外部参照位点的平行分析，从而避免了DNA浓度不可避免的偏差问题。本文介绍的TaqMan检测方法的简便性和可重复性应有助于其在分子诊断实验室的建立。然而，基于SMN区域的复杂性和SMA的临床变异性，SMN基因拷贝数定量分析的适用性需要了解其方法及局限性。因此，在每个病例中都应仔细考虑后，才进行SMN1和SMN2基因拷贝数的测定。
