Detection of Spinal Muscular Atrophy Using a Duplexed Real-Time PCR Approach With Locked Nucleic Acid-Modified Primers.
DNA Primers
/ chemistry
Double-Blind Method
Dried Blood Spot Testing
Genotype
Humans
Infant, Newborn
Muscular Atrophy, Spinal
/ diagnosis
Oligonucleotides
/ chemistry
Prospective Studies
Real-Time Polymerase Chain Reaction
/ methods
Sensitivity and Specificity
Survival of Motor Neuron 1 Protein
/ genetics
Dried blood spot
Locked nucleic acid
Newborn screening
Real-time PCR
Spinal muscular atrophy
Survival motor neuron 1
Journal
Annals of laboratory medicine
ISSN: 2234-3814
Titre abrégé: Ann Lab Med
Pays: Korea (South)
ID NLM: 101571172
Informations de publication
Date de publication:
01 2021
01 2021
Historique:
received:
21
01
2020
revised:
24
03
2020
accepted:
17
07
2020
entrez:
25
8
2020
pubmed:
25
8
2020
medline:
30
6
2021
Statut:
ppublish
Résumé
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. 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. 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. 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.
Sections du résumé
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.
Identifiants
pubmed: 32829585
doi: 10.3343/alm.2021.41.1.101
pii: alm-2021-41-1-101
pmc: PMC7443528
doi:
Substances chimiques
DNA Primers
0
Oligonucleotides
0
SMN1 protein, human
0
Survival of Motor Neuron 1 Protein
0
locked nucleic acid
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
101-107Références
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