Allele-specific amplification of exon 7 in the survival motor neuron (SMN) genes for molecular diagnosis of spinal muscular atrophy.

There are two highly homologous survival motor neuron (SMN) genes in humans but molecular defects in the SMN1 gene cause spinal muscular atrophy (SMA). More than 90% of SMA patients are shown to have a homozygous deletion of exon 7 in the SMN1 gene. Therefore, a simple test for exon 7 deletion would be very useful in the molecular diagnosis of SMA. However, limited methods are available, and most of these methods utilize expensive instruments and consumables. Here, we describe a simple allele-specific PCR test, which can be performed using standard equipment in DNA laboratories. The principle of the test is based on a single nucleotide difference (C versus T) between the exon 7 of SMN1 and SMN2 genes. Using allele-specific primers, two PCR amplifications are performed for each sample to amplify a 404-bp diagnostic fragment, and consequent electrophoresis of PCR products on agarose gel provides definitive information concerning the exon 7 deletion To rule out false negatives, a 500-bp fragment from the N-acetyltransferase gene was coamplified as an internal control in each test. We have, so far, analyzed 41 SMA samples with our method, and tested the validity of results using an independent restriction fragment length polymorphism (RFLP) method. Genotyping results obtained by both methods were in complete agreement for all of the samples analyzed. Our method can also be used to detect heterozygous deletion of exon 7 in SMN genes, if the relative intensities of the diagnostic and internal control bands are determined.