title: Heteroduplex formation in SMN gene dosage analysis.
abstract: Most spinal muscular atrophy patients lack both copies of SMN1 exon 7 and most 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 an assay to determine copy number of SMN1 exon 7 that utilizes multiplex quantitative polymerase chain reaction (PCR) with DraI digestion to differentiate SMN1 from SMN2. Heteroduplex formation in PCR is a well-described phenomenon. In addition to demonstrating the presence of heteroduplexes by sequence analysis of purified SMN1 bands, we compared the SMN1 signals in various 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 SMN1 signal increased as SMN2 copy number increased despite a constant SMN1 copy number, although not all pairwise comparisons showed a statistically significant difference in the SMN1 signal. In conclusion, SMN1/SMN2 heteroduplexes 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 should be considered when performing quantitative multiplex PCR.
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104, USA.
authors.forename: S
authors.initials: S
authors.lastname: Ogino
authors.forename: D G
authors.initials: DG
authors.lastname: Leonard
authors.forename: H
authors.initials: H
authors.lastname: Rennert
authors.forename: S
authors.initials: S
authors.lastname: Gao
authors.forename: R B
authors.initials: RB
authors.lastname: Wilson
mesh_terms: D017362:Cyclic AMP Response Element-Binding Protein; D005091:Exons; D017353:Gene Deletion; D018628:Gene Dosage; D005838:Genotype; D020180:Heteroduplex Analysis; D006801:Humans; D009134:Muscular Atrophy, Spinal / Q000235:genetics*; D009419:Nerve Tissue Proteins / Q000235:genetics*; D009692:Nucleic Acid Heteroduplexes / Q000032:analysis*; D016601:RNA-Binding Proteins; D012189:Retrospective Studies; D055532:SMN Complex Proteins; D055533:Survival of Motor Neuron 1 Protein; D055540:Survival of Motor Neuron 2 Protein
chemical_list: D017362:Cyclic AMP Response Element-Binding Protein; D009419:Nerve Tissue Proteins; D009692:Nucleic Acid Heteroduplexes; D016601:RNA-Binding Proteins; D055532:SMN Complex Proteins; C526471:SMN1 protein, human; C526477:SMN2 protein, human; D055533:Survival of Motor Neuron 1 Protein; D055540:Survival of Motor Neuron 2 Protein