Heteroduplex formation in SMN gene dosage analysis

Most spinal muscular atrophy patients lack both copies of SMN1 exon 7 and m ost carriers have only one copy of SMN1 exon 7. We investigated the effect of SMN1/SMN2 heteroduplex formation on SMN gene dosage analysis, which is a n assay to determine copy number of SMN1 exon 7 that utilizes multiplex qua ntitative polymerase chain reaction (PCR) with DraI digestion to differenti ate SMN1 from SMN2. Heteroduplex formation in PCR is a well-described pheno menon. In addition to demonstrating the presence of heteroduplexes by seque nce analysis of purified SMN1 bands, we compared the SMN1 signals in variou s genotype groups (total n = 260) to those in a group lacking SMN2 (n = 13) , and we estimated the relative amounts of SMN1/SMN2 heteroduplexes. The SM N1 signal increased as SMN2 copy number increased despite a constant SMN1 c opy number, although not all pairwise comparisons showed a statistically si gnificant difference in the SMN1 signal. In conclusion, SMN1/SMN2 heterodup lexes form in SMN gene dosage analysis, falsely increasing the SMN1 signal. External controls for SMN gene dosage analysis should be chosen carefully with regard to SMN2 copy number. The effect of heteroduplex formation shoul d be considered when performing quantitative multiplex PCR.