Spinal muscular atrophy (SMA) is a fatal neuromuscular disorder primarily attributed to the homozygous deletion of the survival motor neuron 1 (SMN1) gene, with disease severity closely correlated to the copy number variations (CNV) of SMN2. Conventional methodologies, however, fail to provide a comprehensive gene overview of SMN and are often both time-intensive and costly. In this study, we present a novel one-step MALDI-TOF MS assay for SMA gene testing. To accurately quantify CNV, we incorporated RPPH1 as an internal control alongside normal samples and competing templates targeting SMN1, SMN2, and RPPH1 for multiple corrections. The CNV assay enables precise quantification of exons 7/8 in both SMN1 and SMN2 genes, achieving a kappa value of 0.935 (P < 0.001) when compared with multiple ligation-dependent probe amplification (MLPA) during its development phase. This accuracy was further corroborated in a cohort comprising 78 individuals. To identify patients harboring compound heterozygous mutations or silent carriers, prevalent pathogenic variants along with sequence variants of SMN1 were integrated into our analysis framework; plasmids were constructed for methodological validation purposes. Utilizing these combinatorial assays for SMN detection, we identified one patient exhibiting a compound heterozygous mutation characterized by genotype [0 + 1^d] and another subject presenting genotype [2 + 1], who harbored simultaneous variants of g.27134T > G and g.27706_27707delAT. The CNV assessment combined with pathogenic variants analysis developed through MALDI-TOF MS provides a comprehensive gene profile of SMN within a single analytical run. Given its unparalleled cost-effectiveness and time efficiency, this approach holds significant promise for further application in clinical diagnosis as well as newborn screening for SMA.

中文翻译：

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通过 MALDI-TOF MS 开发和验证用于脊髓性肌萎缩症基因检测的一步 SMN 检测


脊髓性肌萎缩症 （SMA） 是一种致命的神经肌肉疾病，主要归因于存活运动神经元 1 （SMN1） 基因的纯合缺失，疾病严重程度与 SMN2 的拷贝数变异 （CNV） 密切相关。然而，传统方法无法提供 SMN 的全面基因概述，并且通常既耗时又昂贵。在这项研究中，我们提出了一种用于 SMA 基因检测的新型一步法 MALDI-TOF MS 检测。为了准确定量 CNV，我们将 RPPH1 作为内部对照与正常样品和靶向 SMN1、SMN2 和 RPPH1 的竞争模板一起进行多次校正。CNV 测定能够精确定量 SMN1 和 SMN2 基因中的外显子 7/8，与开发阶段的多重连接依赖性探针扩增 （MLPA） 相比，kappa 值为 0.935 （P < 0.001）。这种准确性在由 78 人组成的队列中得到了进一步的证实。为了识别携带复合杂合突变或沉默携带者的患者，将普遍的致病性变异以及 SMN1 的序列变异整合到我们的分析框架中;构建质粒用于方法学验证目的。利用这些组合分析进行 SMN 检测，我们确定了一名表现出以基因型 [0 + 1^d] 为特征的复合杂合突变的患者和另一名表现出基因型 [2 + 1] 的受试者，他们同时携带 g.27134T > G 和 g.27706_27707delAT 的变体。 CNV 评估与通过 MALDI-TOF MS 开发的致病性变异分析相结合，可在单次分析运行中提供 SMN 的全面基因图谱。鉴于其无与伦比的成本效益和时间效率，这种方法在临床诊断以及新生儿 SMA 筛查中具有重要前景。