Ann Lab Med.  2021 Jan;41(1):101-107. 10.3343/alm.2021.41.1.101.

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

• Pan J
• Zhang C
• Teng Y
• Zeng S
• Chen S
• Liang D
• Li Z
• Wu L

• KMID: 2512739
• DOI: http://doi.org/10.3343/alm.2021.41.1.101

Abstract

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.

Keyword

Spinal muscular atrophy; Survival motor neuron 1; Locked nucleic acid; Realtime PCR; Dried blood spot; Newborn screening

Figure

• 

  Fig. 1 Diagram of the binding location of the SMN1, SMN2, and ALB primers and probes. In primers SMN1-F/R, the 3′ end of the forward primer and reverse primer, targeting the c.835-44g and c.840C site, respectively, has been LNA-modified; the modified bases are indicated in red. Abbreviations: SMN, survival motor neuron; ALB, albumin; LNA, locked nucleic acid; F, forward; R, reverse; P, probe.

• 

  Fig. 2 The amplification specificity of seven different SMN1 primer groups detected by real-time PCR using SMA patient DNA as the template. Each group was set up in triplicate. Abbreviations: SMN1, survival motor neuron 1; ALB, albumin; SMA, spinal muscular atrophy; ΔRn, normalized reporter; F, forward; R, reverse.

• 

  Fig. 3 Primer amplification efficiency and real-time PCR amplification curves. (A) The amplification efficiency of primers SMN1-F1/R1 was 88.4% at 60°C. (B) The amplification efficiency of primers SMN1-F/R was 95.2% at 60°C. (C) The SMN1 and ALB amplification curves in normal individual samples overlapped at the threshold. (D) In SMA carrier samples, the SMN1 amplification curve exhibited an approximate one cycle delay compared with the ALB curve at the threshold. (E) In SMA patient samples, only the ALB gene was amplified. Abbreviations: SMN1, survival motor neuron 1; ALB, albumin; SMA, spinal muscular atrophy; F, forward; R, reverse; ΔRn, normalized reporter.

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