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

doi:10.1002/mgg3.141

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.

Highlights

Spinal muscular atrophy (SMA; OMIM #253300) is an early-onset neurodegenerative disease characterized by the loss of a-motor neurons (MNs) in the anterior horn of the spinal cord (Crawford and Pardo, 1996)
We measured the number of survival motor neuron 1 (SMN1) and SMN2 copies in Genomic DNA (gDNA) isolated from cell lines derived from SMA patients as well as from healthy non-SMA subjects using array digital polymerase chain reaction (PCR) (dPCR) (Fig. 1)
SMN1 or SMN2 dPCR was multiplexed with RPPH1 because the copy number of RPPH1 does not vary amongst the human population (Baer et al, 1990)

Summary

Introduction

Spinal muscular atrophy (SMA; OMIM #253300) is an early-onset neurodegenerative disease characterized by the loss of a-motor neurons (MNs) in the anterior horn of the spinal cord (Crawford and Pardo, 1996). This loss of a-MNs is associated with muscle weakness and atrophy. SMA results from the loss or mutation of SMN1 (survival motor neuron 1; OMIM #600354) on chromosome 5q13 (Lefebvre et al, 1995). A large tandem chromosomal duplication has lead to a second SMN2 copy of the gene (OMIM #601627). The capacity of SMN2 copy number to modulate phenotype has been extended to transgenic mouse models (Monani et al, 2000; Hsieh-Li et al, 2000; Michaud et al, 2010)

Methods

Results

Conclusion