High-throughput genetic newborn screening for spinal muscular atrophy by rapid nucleic acid extraction from dried blood spots and 384-well qPCR
High-throughput genetic newborn screening for spinal muscular atrophy by rapid nucleic acid extraction from dried blood spots and 384-well qPCR
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DOI：
10.1038/s41431-019-0476-4
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发表时间：
2020-01-01
影响因子：
5.2
通讯作者：
Becker, Marc
中科院分区：
文献类型：
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作者：
Czibere, Ludwig;Burggraf, Siegfried;Becker, Marc
建立基于核酸的基因新生儿筛查(NBS)分析为筛查脊髓性肌萎缩症(SMA)等遗传编码疾病提供了可能性，最好是在症状出现之前。这样的分析应该很容易扩展到384孔反应，使每天筛选多达2000个样品成为可能。我们开发了一种基于干血斑清理方案和定量(Q)聚合酶链式反应的检测程序，以筛选运动神经元存活基因(SMN1)外显子7的纯合缺失，该基因与95%的SMA患者有关。该设置的性能得到了详细的评估，并在常规样本上进行了测试。我们从干血斑点中清除核酸的方法产生了足够的DNA，用于随后的各种qPCR应用。到目前为止，我们已经应用这种方法在18个月内测试了213,279个样本。30名患者被确认并确认，这意味着纯合子缺失的发生率为1：7109。使用我们的清理方法，可以建立一个快速的工作流程，从干血斑卡中制备核酸。以外显子7缺失为目标，到目前为止还没有报道无效、假阳性或假阴性的结果。这使得能够及时识别疾病，并在大多数情况下在症状出现之前获得最近引入的治疗选择。运营商没有被识别，因此，不存在是否报告它们的问题。
Establishing nucleic acid-based assays for genetic newborn screening (NBS) provides the possibility to screen for genetically encoded diseases like spinal muscular atrophy (SMA), best before the onset of symptoms. Such assays should be easily scalable to 384-well reactions that make the screening of up to 2000 samples per day possible. We developed a test procedure based on a cleanup protocol for dried blood spots and a quantitative (q)PCR to screen for a homozygous deletion of exon 7 of the survival of motor neuron 1 gene (SMN1) that is responsible for >95% of SMA patients. Performance of this setup is evaluated in detail and tested on routine samples. Our cleanup method for nucleic acids from dried blood spots yields enough DNA for diverse subsequent qPCR applications. To date, we have applied this approach to test 213,279 samples within 18 months. Thirty patients were identified and confirmed, implying an incidence of 1:7109 for the homozygous deletion. Using our cleanup method, a rapid workflow could be established to prepare nucleic acids from dried blood spot cards. Targeting the exon 7 deletion, no invalid, false-positive, or false-negative results were reported to date. This allows timely identification of the disease and grants access to the recently introduced treatment options, in most cases before the onset of symptoms. Carriers are not identified, thus, there are no concerns of whether to report them.