The molecular diagnosis of SMA consists of the detection of the absence of exon 7 of the SMN1 gene (Figure 7-3). Although this is a highly repetitive region and there is the almost identical centromeric SMN2 copy of the SMN1 gene, there is an exonic base pair difference that allows distinction of PCR products of SMN1 from those of SMN2 using restriction-site generating PCR (RG-PCR) followed by restriction enzyme digestion. The absence of detectable SMN1 exon 7 in SMA patients is being utilized as a powerful diagnostic test for SMA, with a sensitivity of approximately 94%. Limitations of this diagnostic test are the inability to detect nondeletion mutations of SMN1 and the inability to determine carrier status for SMA.
Since SMA is one of the most common lethal genetic disorders, with a carrier frequency of 1 in 40 to 60, carrier
testing is useful to many families. Carrier detection for the heterozygous state is technically challenging because the SMA region is characterized by the presence of many repeated elements. The SMN gene is present in two almost identical copies, SMN1 and SMN2. However, the SMN2 gene copy number fluctuates: approximately 5% of normal individuals lack the SMN2 copy, whereas many of the more mildly affected SMA patients have three or more copies of SMN2. Thus, a straightforward dosage assay using the SMN2 gene as the internal control would not be reliable, and the copy number of SMN2 affects the efficiency of amplification of the SMN1 exon 7 region. Quantitative PCR assays, using alternative internal controls, are used for the identification of SMA carriers and determine the number of copies of SMN1 and SMN2 genes.28,33
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