Multiplex Droplet Digital PCR Method Applicable to Newborn Screening, Carrier Status, and Assessment of Spinal Muscular Atrophy.
Clin Chem
; 64(12): 1753-1761, 2018 12.
Article
em En
| MEDLINE
| ID: mdl-30352867
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.
Texto completo:
1
Temas:
ECOS
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Aspectos_gerais
Bases de dados:
MEDLINE
Assunto principal:
Atrofia Muscular Espinal
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Proteína 1 de Sobrevivência do Neurônio Motor
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Reação em Cadeia da Polimerase Multiplex
Tipo de estudo:
Diagnostic_studies
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Screening_studies
Limite:
Female
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Humans
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Male
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Newborn
Idioma:
En
Revista:
Clin Chem
Assunto da revista:
QUIMICA CLINICA
Ano de publicação:
2018
Tipo de documento:
Article