Detection of Spinal Muscular Atrophy Using a Duplexed Real-Time PCR Approach With Locked Nucleic Acid-Modified Primers

Background: Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder mainly caused by homozygous deletions that include exon 7 of the survival motor neuron 1 (SMN1) gene. A nearby paralog gene, SMN2, obstructs the specific detection of SMN1. We optimized a duplexed real-time PCR approach using locked nucleic acid (LNA)-modified primers to specifically detect SMN1. Methods: An LNA-modified primer pair with 3' ends targeting SMN1 specific sites c.835-44g and c.840C was designed, and its specificity was examined by real-time PCR and Sanger Sequencing. A duplexed real-time PCR approach for amplifying SMN1 and control gene albumin (ALB) was developed. A randomized double-blind trial with 97 fresh peripheral blood samples and 25 dried blood spots (DBS) was conducted to evaluate the clinical efficacy of the duplexed approach. This new approach was then used to screen 753 newborn DBS. Results: The LNA-modified primers exhibited enhanced specificity and 6.8% increased efficiency for SMN1 amplification, compared with conventional primers. After stabilizing the SMN1 test by optimizing the duplexed real-time PCR approach, a clinical trial validated that the sensitivity and specificity of our new approach for detecting SMA patients and carriers was 100%. Using this new approach, 15 of the screened 753 newborns were identified as carriers via DBS, while the rest were identified as normal individuals. These data reveal a carrier rate of 1.99% in Hunan province, South Central China. Conclusions: We have developed a novel, specific SMN1 detection approach utilizing real-time PCR with LNA-modified primers. which could be applied to both prenatal carrier and newborn screening.

Automated summary

A novel SMN1 detection approach using LNA-modified primers was developed, which showed enhanced specificity and efficiency compared to conventional primers. Clinical trials validated the 100% sensitivity and specificity of this new approach for detecting SMA patients and carriers. Screening results revealed a carrier rate of 1.99% in Hunan province, South Central China.