PDF(Pubmed)
Abstract：
Spinal muscular atrophy (SMA) is an intractable neuromuscular disorder primarily caused by homozygous deletions in exon 7 of the SMN1 gene. Early diagnosis and prompt treatment of patients with SMA have a significant impact on prognosis, and several therapies have recently been developed. Current SMA screening tests require a significant turnaround time to identify patients with suspected SMA, due both to the interval between the birth of a newborn and the collection of blood for newborn mass screening and the difficulty in distinguishing between SMN1 and SMN2, a paralog gene that requires testing in specialized laboratories. The aim of this study was therefore to develop a novel SMA screening assay that can be rapidly performed in ordinary hospitals and clinics to overcome these issues. We designed over 100 combinations of forward and reverse primers with 3\' ends targeting SMN1-specific sites around exon 7, and evaluated their specificity and amplification efficiency by quantitative PCR to identify the best primer pair. Furthermore, we performed a single-stranded tag hybridization assay after PCR. To evaluate the accuracy and practicality of the newly developed assay, we analyzed saliva specimens from five patients with SMA and two SMA carriers collected in an outpatient clinic and DNA specimens from three patients with SMA and four SMA carriers from a biobank, together with those from healthy individuals. DNA and raw saliva specimens from all patients with SMA demonstrated a biallelic loss of SMN1, whereas those from carriers and healthy individuals did not. The results of 50 independent experiments were consistent for all samples. The assay could be completed within one hour. This simple and convenient new screening tool has the potential to allow patients with SMA to receive disease-modifying therapies within a shorter timeframe.
摘要：
脊髓性肌萎缩症（SMA）是一种主要由SMN1基因外显子7纯合缺失引起的难治性神经肌肉疾病。早期诊断和及时治疗对SMA患者的预后有显著影响,最近开发了几种疗法。当前的SMA筛查测试需要大量的周转时间来识别疑似SMA的患者。由于新生儿出生和收集血液进行新生儿大规模筛查之间的间隔以及难以区分SMN1和SMN2，SMN2是一种需要在专门实验室进行测试的同源基因。因此，这项研究的目的是开发一种新型的SMA筛选测定法，可以在普通医院和诊所中快速进行以克服这些问题。我们设计了100多个正向和反向引物组合，其3'末端靶向外显子7周围的SMN1特异性位点，并通过定量PCR评估其特异性和扩增效率，以确定最佳引物对。此外，我们在PCR后进行了单链标签杂交分析.为了评估新开发的检测方法的准确性和实用性，我们分析了来自门诊诊所收集的5名SMA患者和2名SMA携带者的唾液标本，以及来自生物样本库的3名SMA患者和4名SMA携带者的DNA标本，以及那些来自健康个体的人。来自所有SMA患者的DNA和原始唾液标本均显示SMN1的双等位基因丢失，而来自携带者和健康个体的样本则没有。50个独立实验的结果对于所有样品是一致的。该测定可以在一小时内完成。这种简单方便的新筛查工具有可能使SMA患者在更短的时间内接受疾病改善治疗。
