Strunk Annuska, Abbes Andre, Stuitje Antoine R, Hettinga Chris, Sepers Eline M, Snetselaar Reinier, Schouten Jan, Asselman Fay-Lynn, Cuppen Inge, Lemmink Henny, van der Pol W Ludo, Engel Henk
Department of Clinical Chemistry and Neonatal Screening, Isala Hospital, Dokter van Heesweg 2, 8025 AB Zwolle, The Netherlands.
MRC-Holland, Willem Schoutenstraat 1, 1057 DL Amsterdam, The Netherlands.
Int J Neonatal Screen. 2019 May 15;5(2):21. doi: 10.3390/ijns5020021. eCollection 2019 Jun.
Spinal muscular atrophy (SMA) is one of the leading genetic causes of infant mortality with an incidence of 1:10,000. The recently-introduced antisense oligonucleotide treatment improves the outcome of this disease, in particular when applied at an early stage of progression. The genetic cause of SMA is, in >95% of cases, a homozygous deletion of the survival motor neuron 1 () gene, which makes the low-cost detection of SMA cases as part of newborn screening programs feasible. We developed and validated a new SALSA MC002 melting curve assay that detects the absence of the exon 7 DNA sequence without detecting asymptomatic carriers and reliably discriminates from its genetic homolog using crude extracts from newborn screening cards. Melting curve analysis shows peaks specific for both the gene and the disease modifying homolog. The detection of the homolog, of which the only clinically relevant difference from the gene is a single nucleotide in exon 7, was only used to confirm a correct reaction in samples that lacked the gene, and not for quantification. We retrieved 47 DBS samples from children with genetically-confirmed SMA, after informed consent from parents, and 375 controls from the national archive of the Dutch National Institute for Public Health and the Environment (RIVM). The assay correctly identified all anonymized and randomized SMA and control samples (i.e., sensitivity and specificity of 100%), without the detection of carriers, on the three most commonly-used PCR platforms with melting curve analysis. This test's concordance with the second-tier 'golden standard' P021 SMA MLPA test was 100%. Using the new P021-B1 version, crude extracts from DBS cards could also be used to determine the copy number of SMA patients with a high level of accuracy. The MC002 test showed the feasibility and accuracy of SMA screening in a neonatal screening program.
脊髓性肌萎缩症（SMA）是导致婴儿死亡的主要遗传病因之一，发病率为1:10000。最近引入的反义寡核苷酸疗法改善了这种疾病的治疗效果，尤其是在疾病进展早期应用时。在超过95%的病例中，SMA的遗传病因是生存运动神经元1（SMN1）基因的纯合缺失，这使得作为新生儿筛查项目一部分的SMA病例低成本检测成为可能。我们开发并验证了一种新的SALSA MC002熔解曲线分析方法，该方法可检测外显子7 DNA序列的缺失，而不检测无症状携带者，并能使用新生儿筛查卡片的粗提物可靠地将SMN1与其遗传同源物SMN2区分开来。熔解曲线分析显示了针对SMN1基因和疾病修饰同源物SMN2的特异性峰。SMN2同源物与SMN1基因在临床上唯一相关的差异是外显子7中的一个单核苷酸，其检测仅用于确认缺乏SMN1基因的样本中的正确反应，而不是用于SMN2定量。在获得家长知情同意后，我们从基因确诊的SMA患儿中获取了47份干血斑样本，并从荷兰国家公共卫生和环境研究所（RIVM）的国家档案中获取了375份对照样本。在三种最常用的带有熔解曲线分析的PCR平台上，该分析方法正确识别了所有匿名和随机分组的SMA及对照样本（即灵敏度和特异性均为100%），且未检测到携带者。该检测与二线“金标准”P021 SMA MLPA检测的一致性为100%。使用新的P021 - B1版本，干血斑卡片的粗提物也可用于高精度地确定SMA患者的SMN1拷贝数。MC002检测显示了在新生儿筛查项目中进行SMA筛查的可行性和准确性。
