Development and validation of a 4-color multiplexing spinal muscular atrophy (SMA) genotyping assay on a novel integrated digital PCR instrument.
Journal
Scientific reports
ISSN: 2045-2322
Titre abrégé: Sci Rep
Pays: England
ID NLM: 101563288
Informations de publication
Date de publication:
16 11 2020
16 11 2020
Historique:
received:
05
09
2020
accepted:
02
11
2020
entrez:
17
11
2020
pubmed:
18
11
2020
medline:
12
3
2021
Statut:
epublish
Résumé
Digital 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.
Identifiants
pubmed: 33199817
doi: 10.1038/s41598-020-76893-7
pii: 10.1038/s41598-020-76893-7
pmc: PMC7670453
doi:
Substances chimiques
SMN1 protein, human
0
SMN2 protein, human
0
Survival of Motor Neuron 1 Protein
0
Survival of Motor Neuron 2 Protein
0
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
19892Subventions
Organisme : NIH HHS
ID : R44OD023028
Pays : United States
Organisme : NIH HHS
ID : P20GM103464
Pays : United States
Références
J Child Neurol. 2016 Jun;31(7):899-906
pubmed: 26823478
Genet Med. 2020 May;22(5):945-953
pubmed: 32066871
Am J Hum Genet. 1997 Jul;61(1):9-15
pubmed: 9245977
BMC Bioinformatics. 2012 Jun 18;13:134
pubmed: 22708584
Am J Hum Genet. 2002 Feb;70(2):358-68
pubmed: 11791208
Neurogenetics. 2007 Nov;8(4):271-8
pubmed: 17647030
J Pediatr. 2017 Nov;190:124-129.e1
pubmed: 28711173
J Neurosci. 2015 Jun 10;35(23):8691-700
pubmed: 26063904
PLoS One. 2019 Jul 24;14(7):e0220211
pubmed: 31339938
Lancet. 1986 Feb 8;1(8476):307-10
pubmed: 2868172
Front Genet. 2019 Dec 17;10:1255
pubmed: 31921298
Neuromuscul Disord. 2017 May;27(5):439-446
pubmed: 28284873
Mol Genet Genomic Med. 2015 Jul;3(4):248-57
pubmed: 26247043
Br J Clin Pharmacol. 2019 Jan;85(1):181-193
pubmed: 30302786
Hum Mol Genet. 2017 Jul 15;26(14):2768-2780
pubmed: 28460014
J Neuromuscul Dis. 2020;7(2):97-100
pubmed: 32007960
Sci Rep. 2019 Dec 20;9(1):19606
pubmed: 31862911
Genet Med. 2007 Apr;9(4):241-8
pubmed: 17438389
Anal Chem. 2018 Jan 2;90(1):801-806
pubmed: 29190072
Proc Natl Acad Sci U S A. 1999 Aug 3;96(16):9236-41
pubmed: 10430926
Clin Chem. 2018 Dec;64(12):1753-1761
pubmed: 30352867
Lancet. 2017 Dec 17;388(10063):3017-3026
pubmed: 27939059
Cell. 1995 Jan 13;80(1):155-65
pubmed: 7813012
N Engl J Med. 2017 Nov 2;377(18):1713-1722
pubmed: 29091557
Genet Med. 2018 Jun;20(6):608-613
pubmed: 29758563
Hum Mol Genet. 1999 Jul;8(7):1177-83
pubmed: 10369862
BJOG. 2002 Nov;109(11):1244-9
pubmed: 12452462
Clin Chem. 2018 Dec;64(12):1732-1742
pubmed: 30237149
J Med Genet. 1978 Dec;15(6):409-13
pubmed: 745211
Neurol Clin. 2015 Nov;33(4):831-46
pubmed: 26515624
J Mol Diagn. 2017 Sep;19(5):755-765
pubmed: 28826609
Proc Natl Acad Sci U S A. 2007 Feb 27;104(9):3426-31
pubmed: 17307868
Biotechniques. 1992 Sep;13(3):444-9
pubmed: 1389177
Neuromuscul Disord. 2006 Dec;16(12):830-8
pubmed: 17049859
Front Mol Biosci. 2016 Mar 10;3:7
pubmed: 27014701
Nucleic Acids Res. 1990 Jan 11;18(1):97-103
pubmed: 2308839
Genet Med. 2011 Jul;13(7):686-94
pubmed: 21673580
Genome Res. 2002 Apr;12(4):656-64
pubmed: 11932250
Neurobiol Dis. 1996 Apr;3(2):97-110
pubmed: 9173917
Neurology. 2016 Mar 8;86(10):890-7
pubmed: 26865511
Genet Med. 2014 Feb;16(2):149-56
pubmed: 23788250
Genet Test Mol Biomarkers. 2011 Sep;15(9):587-94
pubmed: 21548796
Proc Natl Acad Sci U S A. 1999 May 25;96(11):6307-11
pubmed: 10339583
J Neuromuscul Dis. 2018;5(2):145-158
pubmed: 29614695
Biomol Detect Quantif. 2016 Aug 08;10:9-14
pubmed: 27990344