SMN1 and SMN2 copy numbers in cell lines derived from patients with spinal muscular atrophy as measured by array digital PCR

Deborah L Stabley, Ashlee W Harris, Jennifer Holbrook, Nicholas J Chubbs, Kevin W Lozo, Thomas O Crawford, Kathryn J Swoboda, Vicky L Funanage, Wenlan Wang, William Mackenzie, Mena Scavina, Katia Sol-Church, Matthew E R Butchbach, Deborah L Stabley, Ashlee W Harris, Jennifer Holbrook, Nicholas J Chubbs, Kevin W Lozo, Thomas O Crawford, Kathryn J Swoboda, Vicky L Funanage, Wenlan Wang, William Mackenzie, Mena Scavina, Katia Sol-Church, Matthew E R Butchbach

Abstract

Proximal spinal muscular atrophy (SMA) 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 mutation of survival motor neuron 1 (SMN1). In the human genome, a large duplication of the SMN-containing region gives rise to a second copy of this gene (SMN2) that is distinguishable by a single nucleotide change in exon 7. Within the SMA population, there is substantial variation in SMN2 copy number; in general, those individuals with SMA who have a high SMN2 copy number have a milder disease. Because SMN2 functions as a disease modifier, its accurate copy number determination may have clinical relevance. In this study, we describe the development of an assay to assess SMN1 and SMN2 copy numbers in DNA samples using an array-based digital PCR (dPCR) system. This dPCR assay 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 copy number and disease severity. Array dPCR is a practical technique to determine, accurately and reliably, SMN1 and SMN2 copy numbers from SMA samples.

Keywords: Array digital PCR; SMN1; SMN2; copy number; copy number variation; spinal muscular atrophy.

Figures

Figure 1
Figure 1
Workflow for SMN1/SMN2 copy number assays, using the QuantStudio 3D array dPCR system.
Figure 2
Figure 2
Comparison of SMN2 copy number in SMA samples determined by qPCR to that by array dPCR. The dashed line represents the linear relationship between SMN2 copy number determined by TaqMan™ qPCR (Gómez-Curet et al., 2007) and that determined by array dPCR.
Figure 3
Figure 3
SMN2 copy number in SMA samples. (A) Distribution of SMN2 copy number in the SMA patient samples (n = 60). (B) Relationship between SMN2 copy number and disease severity in SMN1-deleted SMA samples (n = 59). Each bar represents a clinical grade of SMA. The distribution of SMN2 copy numbers (1 SMN2, red; 2 SMN2, orange; 3 SMN2, yellow and 4 SMN2, green) within each clinical grade is shown within each bar.
Figure 4
Figure 4
SMN1 and SMN2 copy numbers in non-SMA samples. Each bar represents a copy number for SMN1 in the cohort of non-SMA samples (n = 40). The distribution of SMN2 copy numbers (0 SMN2, purple; 1 SMN2, red; 2 SMN2, orange; 3 SMN2, yellow and 5 SMN2, green) within each SMN1 copy number is shown within each bar. None of the samples in our cohort contained 4 copies of SMN2.

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