Departments of Laboratory Medicine and Pathology.
Medical Genetics.
Clin Chem. 2018 Dec;64(12):1753-1761. doi: 10.1373/clinchem.2018.293712. Epub 2018 Oct 23.
Spinal muscular atrophy (SMA) is a progressive neuromuscular disorder with neuronal degeneration leading to muscular atrophy and respiratory failure. SMA is frequently caused by homozygous deletions that include exon 7 of the survival motor neuron gene , and its clinical course is influenced by the copy number of a nearby 5q paralog, . Multiple ligation probe amplification (MLPA) and real-time quantitative PCR (qPCR) can detect deletions. Yet, qPCR needs normalization or standard curves, and MLPA demands DNA concentrations above those obtainable from dried blood spots (DBSs). We developed a multiplex, droplet digital PCR (ddPCR) method for the simultaneous detection of deletions and copy number variation in DBS and other tissues. An Sanger sequencing process for DBS was also developed.
, , and concentrations were simultaneously measured with a Bio-Rad AutoDG and QX200 ddPCR system. A total of 1530 DBSs and 12 SMA patients were tested.
Population studies confirmed 1 to 5 exon 7 copies detected in unaffected specimens, whereas patients with SMA revealed 0 copies. Intraassay and interassay imprecisions were <7.1% CV for individuals with ≥1 copies. Testing 12 SMA-positive samples resulted in 100% sensitivity and specificity.
This ddPCR method is sensitive, specific, and applicable to newborn screening and carrier status determination for SMA. It can also be incorporated with a parallel ddPCR T-cell excision circles assay for severe combined immunodeficiencies.
脊髓性肌萎缩症（SMA）是一种进行性神经肌肉疾病，神经元退化导致肌肉萎缩和呼吸衰竭。SMA 通常由包括生存运动神经元基因外显子 7 的纯合缺失引起，其临床过程受附近 5q 旁系同源物拷贝数的影响。多重连接探针扩增（MLPA）和实时定量 PCR（qPCR）可检测缺失。然而，qPCR 需要归一化或标准曲线，而 MLPA 需要从干血斑（DBS）中获得的高于 DNA 浓度。我们开发了一种多重、液滴数字 PCR（ddPCR）方法，用于同时检测 DBS 和其他组织中的缺失和拷贝数变异。还开发了用于 DBS 的 Sanger 测序过程。
使用 Bio-Rad AutoDG 和 QX200 ddPCR 系统同时测量 、 和 浓度。共检测了 1530 个 DBS 和 12 个 SMA 患者。
人群研究证实，未受影响的标本中检测到 1 到 5 个外显子 7 拷贝，而 SMA 患者则检测到 0 拷贝。个体 拷贝数≥1 时，室内和室间精密度均<7.1%CV。检测 12 个 SMA 阳性样本的灵敏度和特异性均为 100%。
该 ddPCR 方法灵敏、特异，适用于 SMA 的新生儿筛查和携带者状态确定。它还可以与并行 ddPCR T 细胞切除循环检测严重联合免疫缺陷症结合使用。
