Abstract
Spinal muscular atrophy (SMA) is one of the most common and severe genetic diseases. SMA carrier screening is an effective way to identify couples at risk of having affected children. Next-generation sequencing (NGS)-based expanded carrier screening could detect SMN1 gene copy number without extra experiment and with high cost performance. However, its performance has not been fully evaluated. Here we conducted a systematic comparative study to evaluate the performance of three common methods. 478 samples were analyzed with multiplex ligation probe amplification (MLPA), real-time quantitative polymerase chain reaction (qPCR) and NGS, simultaneously. Taking MLPA-based results as the reference, for 0 copy, 1 copy and ≥ 2 copy SMN1 analysis with NGS, the sensitivity, specificity and precision were all 100%. Using qPCR method, the sensitivity was 100%, 97.52% and 94.30%, respectively; 98.63%, 95.48% and 100% for specificity; and 72.72%, 88.72% and 100% for precision. NGS repeatability was higher than that of qPCR. Moreover, among three methods, NGS had the lowest retest rate. Thus, NGS is a relatively more reliable method for SMN1 gene copy number detection. In expanded carrier screening, compared with the combination of multiple methods, NGS method could reduce the test cost and simplify the screening process.
Highlights
Spinal muscular atrophy (SMA) is a common neuromuscular disorder
With multiplex ligation probe amplification (MLPA) method, 16 of the 478 samples were classified as homozygous deletion (0 copy of survival motor neuron 1 (SMN1)), 133 as heterozygous deletion (1 copy of SMN1), and 329 as normal (≥ 2 copies of SMN1); these results were consistent with the previous molecular diagnostic results
The MLPA assay is generally accepted as the gold standard for the molecular analysis of SMA for its high sensitivity and specificity, and it was selected for the validation of the quantitative polymerase chain reaction (qPCR) and Next-generation sequencing (NGS) methods in this study
Summary
Spinal muscular atrophy (SMA) is a common neuromuscular disorder. It is attributed to the degeneration of spinal and brainstem motor neurons, leading to progressive muscle weakness and atrophy and even respiratory failure and death. SMA is divided into four clinical subtypes according to the onset age and clinical manifestations[3] All these forms are caused by mutations in the survival motor neuron 1 (SMN1) gene. The frequently used methods for SMA carrier screening are real-time quantitative polymerase chain reaction (qPCR)[11,12,13,14], denaturing high-performance liquid. We selected the frequently used qPCR method and the promising NGS approach and investigated their performance systematically for SMA detection. These methods were carried out in accordance with relevant guidelines and regulations
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