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

Lingxia Jiang, Robert Lin, Steve Gallagher, Andrew Zayac, Matthew E R Butchbach, Paul Hung, Lingxia Jiang, Robert Lin, Steve Gallagher, Andrew Zayac, Matthew E R Butchbach, Paul Hung

Abstract

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.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Components of the Combinati Absolute Q Digital PCR System with a 16-sample microfluidic array partitioning (MAP) plate. The instrument integrates thermal systems for PCR, pneumatic systems for reagent digitization and optical systems for raw data acquisition.
Figure 2
Figure 2
Schematic representation of the 4-color multiplex assay design showing the positions of the 4 probes. Arrows represent forward and reverse primers of each assay. The bars connecting two circles represent probes while the color-filled circles represent different fluorophores and the black circles represent dark quenchers. The dashed lines (//) indicate discontinuous sequence.
Figure 3
Figure 3
Comparison of the assay workflow for the QuantStudio 3D dPCR System (A; ) and the Combinati Absolute Q dPCR System (B). Panel A is reproduced from with permission from the authors (2015) in accordance with Creative Commons license CC BY 3.0.
Figure 4
Figure 4
Representative partition image (A) and scatter plots (B–D) of the SMA 4-color multiplexing dPCR assay using NA03815 genomic DNA as the sample. The scatter plots are shown for SMN1 exon 7 (B; FAM), SMN2 exon 7 (C; VIC) and total SMN intron 1 (D; TYE665), all relative to the reference gene RPPH1 (TAMRA). The analysis software subtracted the pre-PCR image intensities from the post-PCR image intensities so as to remove fluorescent signals which did not exhibit amplification behavior, thereby eliminating false positive signals. If the pre-PCR partitions were brighter than the post-PCR partitions, then the analysis software would record negative fluorescent units. These negative units, however, did not impact the copy number quantification results.
Figure 5
Figure 5
Repeatability of the Absolute Q dPCR assays for SMA genotyping. The control DNA samples (n = 10) from Coriell Cell Repositories were assayed for SMN1 (A), SMN2 (B) and total SMN (C) copy numbers. Each sample was assayed in triplicate. The intra-assay precision, measured by %CV, for each assay was listed below each sample on the plots. The intra-assay precision measurements were not calculated (N/C) for those samples with a copy number less than 1.00.
Figure 6
Figure 6
Agreement between the QuantStudio 3D and the Absolute Q dPCR assays. (A–C) Scatterplots for SMN1 (A), SMN2 (B) and total SMN (C) copy numbers measured with the QuantStudio 3D and the Absolute Q dPCR assays. The line of equality for each set of assays is shown as a dashed line in each plot. (D–F) Bland Altman difference plots for SMN1 (D), SMN2 (E) and total SMN (F) assays. For each assay, the bias is shown as a solid blue line that is labeled as the mean and the limits of agreement defined and labeled as the mean ± 1.96SD (standard deviation).

References

    1. Crawford TO, Pardo CA. The neurobiology of childhood spinal muscular atrophy. Neurobiol. Dis. 1996;3:97–110. doi: 10.1006/nbdi.1996.0010.
    1. Tisdale S, Pellizzoni L. Disease mechanisms and therapeutic approaches in spinal muscular atrophy. J. Neurosci. 2015;35:8691–8700. doi: 10.1523/JNEUROSCI.0417-15.2015.
    1. Pearn J. Incidence, prevalence and gene frequency studies of chronic childhood spinal muscular atrophy. J. Med. Genet. 1978;15:409–413. doi: 10.1136/jmg.15.6.409.
    1. Cuscó I, et al. Prenatal diagnosis for risk of spinal muscular atrophy. Br. J. Obstet. Gynaecol. 2002;109:1244–1249. doi: 10.1046/j.1471-0528.2002.02083.x.
    1. Lefebvre S, et al. Identification and characterization of a spinal muscular atrophy-determining gene. Cell. 1995;80:155–165. doi: 10.1016/0092-8674(95)90460-3.
    1. Lorson CL, Hahnen E, Androphy EJ, Wirth B. A single nucleotide in the SMN gene regulates splicing and is responsible for spinal muscular atrophy. Proc. Natl. Acad. Sci. USA. 1999;96:6307–6311. doi: 10.1073/pnas.96.11.6307.
    1. Monani UR, et al. A single nucleotide difference that alters splicing patterns distinguishes the SMA gene SMN1 from the copy gene SMN2. Hum. Mol. Genet. 1999;8:1177–1183. doi: 10.1093/hmg/8.7.1177.
    1. Kolb SJ, Kissel JT. Spinal muscular atrophy. Neurol. Clin. 2015;33:831–846. doi: 10.1016/j.ncl.2015.07.004.
    1. Butchbach MER. Copy number variations in the survival motor neuron genes: implications for spinal muscular atrophy and other neurodegenerative diseases. Front. Mol. Biosci. 2016;3:7. doi: 10.3389/fmolb.2016.00007.
    1. Chiriboga CA, et al. Results from a phase I study of nusinersen (ISIS-SMNrx) in children with spinal muscular atrophy. Neurology. 2016;86:890–897. doi: 10.1212/WNL.0000000000002445.
    1. Finkel RS, et al. Treatment of infantile-onset spinal muscular atrophy with nusinersen: a phase 2, open-label, dose-escalation study. Lancet. 2016;388:3017–3026. doi: 10.1016/S0140-6736(16)31408-8.
    1. Haché M, et al. Intrathecal injections in children with spinal muscular atrophy: nusinersen clinical trial experience. J. Child Neurol. 2016;31:899–906. doi: 10.1177/0883073815627882.
    1. Mendell JR, et al. Single-dose gene-replacement therapy for spinal muscular atrophy. N. Engl. J. Med. 2017;377:1713–1722. doi: 10.1056/NEJMoa1706198.
    1. Sturm S, et al. A phase 1 healthy male volunteer single escalating dose study of the pharmacokinetics and pharmacodynamics of risdiplam (RG7916, RO7034067), a SMN2 splicing modifier. Br. J. Clin. Pharmacol. 2019;85:181–193. doi: 10.1111/bcp.13786.
    1. Glascock J, et al. Treatment algorithm for infants diagnosed with spinal muscular atrophy through newborn screening. J. Neuromuscul. Dis. 2018;5:145–158. doi: 10.3233/JND-180304.
    1. Glascock J, et al. Revised recommendations for the treatment of infants diagnosed with spinal muscular atrophy via newborn screening who have 4 copies of SMN2. J. Neuromuscul. Dis. 2020;7:97–100. doi: 10.3233/JND-190468.
    1. Feldkötter M, Schwarzer V, Wirth R, Wienker TF, Wirth B. Quantitative analyses of SMN1 and SMN2 based on real-time LightCycler PCR: fast and highly reliable carrier testing and prediction of severity of spinal muscular atrophy. Am. J. Hum. Genet. 2002;70:358–368. doi: 10.1086/338627.
    1. Arkblad EL, et al. Multiplex ligation-dependent probe amplification improves diagnostics in spinal muscular atrophy. Neuromuscul. Disord. 2006;16:830–838. doi: 10.1016/j.nmd.2006.08.011.
    1. Huang CH, et al. Copy number analysis of survival motor neuron genes by multiplex ligation-dependent probe amplification. Genet. Med. 2007;9:241–248. doi: 10.1097/GIM.0b013e31803d35bc.
    1. Gómez-Curet I, et al. Robust quantification of the SMN gene copy number by real-time Taqman PCR. Neurogenetics. 2007;8:271–278. doi: 10.1007/s10048-007-0093-1.
    1. Alías L, et al. Accuracy of marker analysis, quantitative real-time polymerase chain reaction and multiple ligation-dependent probe amplification to determine SMN2 copy number in patients with spinal muscular atrophy. Genet. Test. Mol. Biomarkers. 2011;15:587–594. doi: 10.1089/gtmb.2010.0253.
    1. Prior TW, Nagan N, Sugarman EA, Batish SD, Braastad C. Technical standards and guidelines for spinal muscular atrophy testing. Genet. Med. 2011;13:686–694. doi: 10.1097/GIM.0b013e318220d523.
    1. Chen X, et al. Spinal muscular atrophy diagnosis and carrier screening from genome sequencing data. Genet. Med. 2020;22:945–953. doi: 10.1038/s41436-020-0754-0.
    1. Butchbach MER. Applicability of digital PCR to the investigation of pediatric-onset genetic disorders. Biomol. Detect. Quant. 2016;10:9–14.
    1. Sykes PJ, et al. Quantitation of targets for PCR by use of limiting dilution. Biotechniques. 1992;13:444–449.
    1. Vogelstein B, Kinzler KW. Digital PCR. Proc. Natl. Acad. Sci. USA. 1999;96:9236–9241. doi: 10.1073/pnas.96.16.9236.
    1. Stabley DL, et al. SMN1 And SMN2 copy numbers in cell lines derived from patients with spinal muscular atrophy as measured by array digital PCR. Mol. Genet. Genom. Med. 2015;3:248–257. doi: 10.1002/mgg3.141.
    1. Vidal-Folch N, et al. Multiplex droplet digital PCR method applicable to newborn screening, carrier status and assessment of spinal muscular atrophy. Clin. Chem. 2018;64:1753–1761. doi: 10.1373/clinchem.2018.293712.
    1. Dueck ME, et al. Precision cancer monitoring using a novel, fully integrated, microfluidic array partitioning digital PCR platform. Sci. Rep. 2019;9:19606. doi: 10.1038/s41598-019-55872-7.
    1. Baer M, Nilsen TW, Costigan C, Altman S. Structure and transcription of a human gene for H1 RNA, the RNA component of human RNase P. Nucleic Acids Res. 1990;18:97. doi: 10.1093/nar/18.1.97.
    1. Stabley DL, et al. Establishing a reference dataset for the authentication of spinal muscular atrophy cell lines using STR profiling and digital PCR. Neuromuscul. Disord. 2017;27:439–446. doi: 10.1016/j.nmd.2017.02.002.
    1. Kent WJ. BLAT: the BLAST-like alignment tool. Genome Res. 2002;12:656–664. doi: 10.1101/gr.229202.
    1. Vijzelaar R, et al. The frequency of SMN gene variants lacking exon 7 and 8 is highly population dependent. PLoS ONE. 2019;14:E0220211. doi: 10.1371/journal.pone.0220211.
    1. Burghes AHM. When is a deletion not a deletion? When it is converted. Am. J. Hum. Genet. 1997;61:9–15. doi: 10.1086/513913.
    1. Kashima T, Rao N, Manley JL. An intronic element contributes to splicing repression in spinal muscular atrophy. Proc. Natl. Acad. Sci. USA. 2007;104:3426–3431. doi: 10.1073/pnas.0700343104.
    1. Wu X, et al. A-44G transition in SMN2 intron 6 protects patients with spinal muscular atrophy. Hum. Mol. Genet. 2017;26:2768–2780. doi: 10.1093/hmg/ddx166.
    1. Luo M, et al. An ashkenazi jewish SMN1 haplotype specific to duplication alleles improves pan-ethnic carrier screening for spinal muscular atrophy. Genet. Med. 2014;16:149–156. doi: 10.1038/gim.2013.84.
    1. Chien YH, et al. Presymptomatic diagnosis of spinal muscular atrophy through newborn screening. J. Pediatr. 2017;190:124–129. doi: 10.1016/j.jpeds.2017.06.042.
    1. Kraszewski JN, et al. Pilot study of population-based newborn screening for spinal muscular atrophy in New York state. Genet. Med. 2018;20:608–613. doi: 10.1038/gim.2017.152.
    1. Durie D, et al. Quantification of DNA in neonatal dried blood spots by adenine tandem mass spectrometry. Anal. Chem. 2018;90:801–806. doi: 10.1021/acs.analchem.7b03265.
    1. Lin Y, et al. Newborn screening for spinal muscular atrophy in China using DNA mass spectrometry. Front. Genet. 2019;10:1255. doi: 10.3389/fgene.2019.01255.
    1. Vidal-Folch N, et al. A droplet digital PCR method for severe combined immunodeficiency newborn screening. J. Mol. Diagn. 2017;19:755–765. doi: 10.1016/j.jmoldx.2017.05.011.
    1. Milosevic D, et al. Applying standard clinical chemistry assay validation to droplet digital PCR quantitative liquid biopsy testing. Clin. Chem. 2018;64:1732–1742. doi: 10.1373/clinchem.2018.291278.
    1. Ye J, et al. Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction. BMC Bioinform. 2012;13:134. doi: 10.1186/1471-2105-13-134.
    1. Bland JM, Altman DG. Statistical methods for assessing agreement between two methods of clinical measurement. Lancet. 1986;327:307–310. doi: 10.1016/S0140-6736(86)90837-8.

Source: PubMed

Sponsoren und Mitarbeiter

Krankheiten

Drogeninterventionen

3
Abonnieren