Abstract
Purpose: To compare the accuracy of a commercially available MLPA kit with a laboratory developed RT-PCR assay for the detection of SMN1 and SMN2 copy numbers in clinical samples. Methods: We developed and validated a laboratory developed real time PCR based test capable of detecting SMN1 and SMN2 copy numbers in individuals. We also validated an MLPA kit purchased from MRC Holland for the same purpose. We then analyzed a series of 1027 anonymized samples using both technologies. When discrepant results were obtained, each sample was re-analyzed at least twice using both platforms. Results: Five samples did not yield results in either assay. For SMN1 copy number analysis, 2 RT-PCR assays revealed indeterminant results and all 1020 other samples were concordant for SMN1 copy number. There were 9 discrepancies in SMN2 copy number determination mostly due to a variability in MLPA analysis. Conclusion: Both MLPA and RTPCR assays give a reliable estimate of SMN1 copy number and are therefore appropriate for population based carrier screening for Spinal Muscular Atrophy Type 1. The MLPA kit has a low incidence (
Highlights
Spinal Muscular Atrophy Type 1 (SMA-1, OMIM #253300) or Werdnig-Hoffman disease is an autosomal recessive neurodegenerative disorder with an estimated incidence of 1:10,000 live births
We developed and validated a laboratory developed real time PCR based test capable of detecting survivor motor neuron 1 (SMN1) and SMN2 copy numbers in individuals
There were 9 discrepancies in SMN2 copy number determination mostly due to a variability in Multiplex Ligation Dependant Probe Amplification (MLPA) analysis. Both MLPA and RTPCR assays give a reliable estimate of SMN1 copy number and are appropriate for population based carrier screening for Spinal Muscular Atrophy Type 1
Summary
253300) or Werdnig-Hoffman disease is an autosomal recessive neurodegenerative disorder with an estimated incidence of 1:10,000 live births. Ninety-five percent of SMA1 cases are due to homozygous deletions of exon 7 of the survivor motor neuron 1 (SMN1) gene. The identification of carriers of SMA relies on determining the copy number of exon 7 of the SMN1 gene. Individuals with 2 or more copies of exon 7 of SMN1 are considered to be non-carriers of SMA. Some carrier individuals will harbor point mutations or small insertions/deletions in an SMN1 gene and some individuals. Individuals with a single copy of exon 7 of SMN1 gene are carriers of SMA. We compared the performance of these 2 assays in detecting copy numbers of exon 7 of both SMN1 and SMN2 genes. This study determined that both methods are capable of identifying the SMN1 exon 7 copy number and are acceptable for the purposes of carrier screening, but the determination of SMN2 copy number was somewhat more problematic
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