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Detection of SMN1 to SMN2 gene conversion events and partial SMN1 gene deletions using array digital PCR

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Abstract

Proximal spinal muscular atrophy (SMA), a leading genetic cause of infant death worldwide, is an early-onset motor neuron disease characterized by loss of α-motor neurons and associated muscle atrophy. SMA is caused by deletion or other disabling mutations of survival motor neuron 1 (SMN1) but retention of one or more copies of the paralog SMN2. Within the SMA population, there is substantial variation in SMN2 copy number (CN); in general, those individuals with SMA who have a high SMN2 CN have a milder disease. Because SMN2 functions as a disease modifier, its accurate CN determination may have clinical relevance. In this study, we describe the development of array digital PCR (dPCR) to quantify SMN1 and SMN2 CNs in DNA samples using probes that can distinguish the single nucleotide difference between SMN1 and SMN2 in exon 8. This set of dPCR assays can accurately and reliably measure the number of SMN1 and SMN2 copies in DNA samples. In a cohort of SMA patient–derived cell lines, the assay confirmed a strong inverse correlation between SMN2 CN and disease severity. We can detect SMN1–SMN2 gene conversion events in DNA samples by comparing CNs at exon 7 and exon 8. Partial deletions of SMN1 can also be detected with dPCR by comparing CNs at exon 7 or exon 8 with those at intron 1. Array dPCR is a practical technique to determine, accurately and reliably, SMN1 and SMN2 CNs from SMA samples as well as identify gene conversion events and partial deletions of SMN1.

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Abbreviations

CN:

copy number

CV:

coefficient of variation

dPCR:

digital PCR

dsDNA:

double-stranded DNA

ex7:

exon 7

ex8:

exon 8

6FAM:

6-carboxyfluorescein

gDNA:

genomic DNA

in1:

intron 1

LCL:

lymphoblastoid-like cell line

PCR:

polymerase chain reaction

SD:

standard deviation

SMA:

spinal muscular atrophy

SMN:

survival motor neuron

3’-UTR:

3′-untranslated region

VIC:

4,7,2′-trichloro-7′-phenyl-6-carboxyfluorescein

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Funding

This project was supported by funding from the National Institute of General Medical Sciences of the National Institutes of Health (Institutional Development Award (IDeA) Networks of Biomedical Research Excellence; P20GM103446 to M.E.R.B. and K.M.R. and Centers of Biomedical Research Excellence; P20GM103464 to M.E.R.B.) and the Nemours Foundation (M.E.R.B. and K.M.R.). Some of the fibroblast lines used in this study were obtained from the Intellectual and Developmental Disabilities Research Center at the Kennedy Krieger Institute, Baltimore, MD which is supported by the NICHD grant 1U54HD079123-01A1.

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Authors and Affiliations

  1. Nemours Biomolecular Core Laboratory, Nemours Biomedical Research, Nemours Alfred I. duPont Hospital for Children, Wilmington, DE, USA

    Deborah L. Stabley, Jennifer Holbrook & Katherine M. Robbins

  2. Division of Neurology, Nemours Alfred I. duPont Hospital for Children, Wilmington, DE, USA

    Mena Scavina

  3. Department of Neurology, Johns Hopkins University, Baltimore, MD, USA

    Thomas O. Crawford

  4. Department of Pediatrics, Johns Hopkins University, Baltimore, MD, USA

    Thomas O. Crawford

  5. Department of Neurology, Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, USA

    Kathryn J. Swoboda

  6. Center for Applied Clinical Genomics, Nemours Biomedical Research, Nemours Alfred I. duPont Hospital for Children, Wilmington, DE, USA

    Matthew E. R. Butchbach

  7. Department of Biological Sciences, University of Delaware, Newark, DE, USA

    Matthew E. R. Butchbach

  8. Department of Pediatrics, Thomas Jefferson University, Philadelphia, PA, USA

    Matthew E. R. Butchbach

  9. Center for Pediatric Research, Nemours Biomedical Research, Nemours Alfred I. duPont Hospital for Children, 4462 E400 DuPont Experimental Station, 200 Powder Mill Road, Wilmington, DE, 19803, USA

    Matthew E. R. Butchbach

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  1. Deborah L. Stabley
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  2. Jennifer Holbrook
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Contributions

D.L.S., K.M.R., and M.E.R.B. conceived and designed the experiments; D.L.S. and J.H. performed the experiments; D.L.S. and M.E.R.B. analyzed data; T.O.C., M.S., and K.J.S. provided key reagents; D.L.S. and M.E.R.B. wrote the first draft of the manuscript; D.L.S., J.H., M.S., T.O.C., K.J.S., K.M.R., and M.E.R.B. edited the manuscript.

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Correspondence to Matthew E. R. Butchbach.

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All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or research committees (Nemours Institutional Review Board, #764456) and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. This article does not contain any studies with animals performed by any of the authors.

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Stabley, D.L., Holbrook, J., Scavina, M. et al. Detection of SMN1 to SMN2 gene conversion events and partial SMN1 gene deletions using array digital PCR. Neurogenetics 22, 53–64 (2021). https://doi.org/10.1007/s10048-020-00630-5

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