scholarly journals Multiplex Droplet Digital PCR Method Applicable to Newborn Screening, Carrier Status, and Assessment of Spinal Muscular Atrophy

2018 ◽  
Vol 64 (12) ◽  
pp. 1753-1761 ◽  
Author(s):  
Noemi Vidal-Folch ◽  
Dimitar Gavrilov ◽  
Kimiyo Raymond ◽  
Piero Rinaldo ◽  
Silvia Tortorelli ◽  
...  
Keyword(s):  
Spinal Muscular Atrophy ◽  
Newborn Screening ◽  
Copy Number ◽  
Neuronal Degeneration ◽  
Muscular Atrophy ◽  
Simultaneous Detection ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Carrier Status ◽  
Survival Motor Neuron Gene

Abstract BACKGROUND Spinal muscular atrophy (SMA) is a progressive neuromuscular disorder with neuronal degeneration leading to muscular atrophy and respiratory failure. SMA is frequently caused by homozygous deletions that include exon 7 of the survival motor neuron gene SMN1, and its clinical course is influenced by the copy number of a nearby 5q SMN1 paralog, SMN2. Multiple ligation probe amplification (MLPA) and real-time quantitative PCR (qPCR) can detect SMN1 deletions. Yet, qPCR needs normalization or standard curves, and MLPA demands DNA concentrations above those obtainable from dried blood spots (DBSs). We developed a multiplex, droplet digital PCR (ddPCR) method for the simultaneous detection of SMN1 deletions and SMN2 copy number variation in DBS and other tissues. An SMN1 Sanger sequencing process for DBS was also developed. METHODS SMN1, SMN2, and RPP30 concentrations were simultaneously measured with a Bio-Rad AutoDG and QX200 ddPCR system. A total of 1530 DBSs and 12 SMA patients were tested. RESULTS Population studies confirmed 1 to 5 SMN1 exon 7 copies detected in unaffected specimens, whereas patients with SMA revealed 0 SMN1 copies. Intraassay and interassay imprecisions were <7.1% CV for individuals with ≥1 SMN1 copies. Testing 12 SMA-positive samples resulted in 100% sensitivity and specificity. CONCLUSIONS This ddPCR method is sensitive, specific, and applicable to newborn screening and carrier status determination for SMA. It can also be incorporated with a parallel ddPCR T-cell excision circles assay for severe combined immunodeficiencies.

scholarly journals Cloning, Characterization, and Copy Number of the Murine Survival Motor Neuron Gene: Homolog of the Spinal Muscular Atrophy-Determining Gene

1997 ◽  
Vol 7 (4) ◽  
pp. 339-352 ◽  
Author(s):  
Christine J. DiDonato ◽  
Xiao-Ning Chen ◽  
David Noya ◽  
Julie R. Korenberg ◽  
Joseph H. Nadeau ◽  
...  
Keyword(s):  
Spinal Muscular Atrophy ◽  
Motor Neuron ◽  
Copy Number ◽  
Muscular Atrophy ◽  
Survival Motor Neuron ◽  
Survival Motor Neuron Gene

Analytical validation of the droplet digital PCR assay for diagnosis of spinal muscular atrophy

2020 ◽  
Vol 510 ◽  
pp. 787-789 ◽  
Author(s):  
Sunggyun Park ◽  
Hyeonah Lee ◽  
Saeam Shin ◽  
Seung-Tae Lee ◽  
Kyung-A Lee ◽  
...  
Keyword(s):  
Spinal Muscular Atrophy ◽  
Muscular Atrophy ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Analytical Validation ◽  
Pcr Assay

scholarly journals Assessment of Spinal Muscular Atrophy Carrier Status by Determining SMN1 Copy Number Using Dried Blood Spots

2020 ◽  
Vol 6 (2) ◽  
pp. 43
Author(s):  
Yogik Onky Silvana Wijaya ◽  
Jamiyan Purevsuren ◽  
Nur Imma Fatimah Harahap ◽  
Emma Tabe Eko Niba ◽  
Yoshihiro Bouike ◽  
...  
Keyword(s):  
Spinal Muscular Atrophy ◽  
Copy Number ◽  
Muscular Atrophy ◽  
Autosomal Recessive Inheritance ◽  
Dried Blood Spots ◽  
Carrier Screening ◽  
Carrier Status ◽  
Essential Information ◽  
Blood Spots ◽  
Incurable Disease

Spinal muscular atrophy (SMA) is a common neuromuscular disease with autosomal recessive inheritance. The disease gene, SMN1, is homozygously deleted in 95% of SMA patients. Although SMA has been an incurable disease, treatment in infancy with newly developed drugs has dramatically improved the disease severity. Thus, there is a strong rationale for newborn and carrier screening for SMA, although implementing SMA carrier screening in the general population is controversial. We previously developed a simple, accurate newborn SMA screening system to detect homozygous SMN1 deletions using dried blood spots (DBS) on filter paper. Here, we modified our previous system to detect the heterozygous deletions of SMN1, which indicates SMA carrier status. The system involves a calibrator-normalized relative quantification method using quantitative nested PCR technology. Our system clearly separated the DBS samples with one SMN1 copy (carrier status with a heterozygous deletion of SMN1) from the DBS samples with two SMN1 copies (non-carrier status with no deletion of SMN1). We also analyzed DBS samples from SMA families, confirmed SMA in the affected children, and determined the carrier status of their parents based on the SMN1 copy number. In conclusion, our system will provide essential information for risk assessment and genetic counseling, at least for SMA families.

scholarly journals Development and validation of a 4-color multiplexing spinal muscular atrophy (SMA) genotyping assay on a novel integrated digital PCR instrument

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Lingxia Jiang ◽  
Robert Lin ◽  
Steve Gallagher ◽  
Andrew Zayac ◽  
Matthew E. R. Butchbach ◽  
...  
Keyword(s):  
Spinal Muscular Atrophy ◽  
Copy Number ◽  
Muscular Atrophy ◽  
Digital Pcr ◽  
Copy Number Variations ◽  
Higher Order ◽  
Clinical Samples ◽  
Internal Reference ◽  
Genotyping Assay ◽  
Copy Numbers

AbstractDigital PCR (dPCR) technology has been proven to be highly sensitive and accurate in detecting copy number variations (CNV). However, a higher-order multiplexing dPCR assay for measuring SMN1 and SMN2 copy numbers in spinal muscular atrophy (SMA) samples has not been reported. Described here is a rapid multiplex SMA dPCR genotyping assay run on a fully integrated dPCR instrument with five optical channels. The hydrolysis probe-based multiplex dPCR assay quantifies SMN1, SMN2, and the total SMN (SMN1 + SMN2) while using RPPH1 gene as an internal reference control. The quadruplex assay was evaluated with characterized control DNA samples and validated with 15 blinded clinical samples from a previously published study. SMN1 and SMN2 copy numbers were completely concordant with previous results for both the control and blinded samples. The dPCR-based SMA copy number determination was accomplished in 90 min with a walk-away workflow identical to real-time quantitative PCR (qPCR). In summary, presented here is a simple higher-order multiplexing solution on a novel digital PCR platform to meet the growing demand for SMA genotyping and prognostics.

scholarly journals Massachusetts’ Findings from Statewide Newborn Screening for Spinal Muscular Atrophy

2021 ◽  
Vol 7 (2) ◽  
pp. 26
Author(s):  
Jaime E. Hale ◽  
Basil T. Darras ◽  
Kathryn J. Swoboda ◽  
Elicia Estrella ◽  
Jin Yun Helen Chen ◽  
...  
Keyword(s):  
Spinal Muscular Atrophy ◽  
Newborn Screening ◽  
New England ◽  
Screening Program ◽  
Muscular Atrophy ◽  
Treatment Options ◽  
Newborn Screening Program ◽  
Treatment Protocols ◽  
Screening Algorithm ◽  
New Treatment

Massachusetts began newborn screening (NBS) for Spinal Muscular Atrophy (SMA) following the availability of new treatment options. The New England Newborn Screening Program developed, validated, and implemented a screening algorithm for the detection of SMA-affected infants who show absent SMN1 Exon 7 by Real-Time™ quantitative PCR (qPCR). We screened 179,467 neonates and identified 9 SMA-affected infants, all of whom were referred to a specialist by day of life 6 (average and median 4 days of life). Another ten SMN1 hybrids were observed but never referred. The nine referred infants who were confirmed to have SMA were entered into treatment protocols. Early data show that some SMA-affected children have remained asymptomatic and are meeting developmental milestones and some have mild to moderate delays. The Massachusetts experience demonstrates that SMA NBS is feasible, can be implemented on a population basis, and helps engage infants for early treatment to maximize benefit.

SMN2 copy number predicts acute or chronic spinal muscular atrophy but does not account for intrafamilial variability in siblings

2005 ◽  
Vol 253 (1) ◽  
pp. 21-25 ◽  
Author(s):  
I. Cuscó ◽  
M. J. Barceló ◽  
R. Rojas–García ◽  
I. Illa ◽  
J. Gámez ◽  
...  
Keyword(s):  
Spinal Muscular Atrophy ◽  
Copy Number ◽  
Muscular Atrophy ◽  
Intrafamilial Variability ◽  
Chronic Spinal Muscular Atrophy
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