Use of a novel Pyrosequencing approach for Survival
Motor Neuron Gene Copy Number quantification
1Department of Biological
Science, University of
Delaware, Newark, DE; 2Nemours Biomedical Research, Alfred
I. duPont
Hospital for Children, Wilmington, DE; 3Department of
Pediatrics,
Thomas Jefferson University, Philadelphia, PA; 4Departments
of
Neurology and Pediatrics, Johns Hopkins University, Baltimore, MD
Spinal
Muscular Atrophy (SMA) is an autosomal recessive
disease caused by mutation or deletion of the telomeric copy of the survival
motor neuron gene 1 (SMN1). The centromeric
copy of the gene, SMN2,
is present in all patients but unable to fully compensate for SMN1. SMN2
has a C→T transition in exon 7 that leads to exon skipping. As a
result, the
majority of transcripts from SMN2 gene lack exon 7.
Thus, SMA results
from insufficient levels of SMN protein in motor neurons. Although SMN1
is the disease-determining gene, the number of SMN2 copies appears to
modulate
SMA clinical phenotype. Determination of SMN copy number is
therefore
important for clinical diagnosis and prognosis. We have developed a
Multiplex
pyrosequencing assay that allows sensitive and quantitative
determination of
SMN copy number. This assay includes the cystic fibrosis
transmembrane
regulator
gene (CFTR) as an internal
standard of known copy number
(2 copies/genome). SMN and CFTR are amplified by
multiplex PCR
using gene-specific and universal primers to achieve equal
amplification
efficiency. The PCR sample is then run in a multiplex pyrosequencing
assay and
data is acquired as peaks in a pyrogram in which peak heights are
directly
proportional to nucleotide incorporation. SMN copy number is
determined by
comparing the peak height at the SNP site (C/T) in the SMN gene to the
peak height at one or more positions that are specific for the internal
standard CFTR. This assay was
validated with DNA samples of known SMN
copy number and will be used to determine SMN copy number of DNA
samples
isolated from SMA and control fibroblasts. The development of this
assay will
allow characterization of SMA cell lines and may also generate a
quantitative
assay that could be used for molecular diagnosis of SMA. Funding for
this
research has been provided by the Howard Hughes Medical Institute
Fellowship
and by grant support: Nemours and NIH 1 P20 RR020173-01.
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