A Duplex Allele-Specific Amplification PCR to Detect SMN1 Deletion Patrı ´cia de Campos Pieri, 1 Jeronimo de Alencar Nogueira, 1 Maria Joaquina Marques-Dias, 2 Bernadete Resende, 3 Chong Ae Kim, 4 Umbertina Conti Reed, 3 and Thelma Suely Okay 1 Spinal muscular atrophy (SMA), the leading genetic cause of death in childhood, is an autosomal recessive neuromuscular disorder characterized by progressive muscle weakness, associated with deletions of the survival motor neuron (SMN) gene identified and mapped to chromosome 5q13. SMN is present in two highly ho- mologous copies (SMN1 and SMN2). In the general population, normal individuals (noncarriers) have at least one telomeric (SMN1) copy, and 5% of them have no copies of SMN2. Approximately 95% of SMA patients carry homologous deletions of SMN1 exon(s) 7 (and 8). SMN1 and SMN2 exons 7 and 8 differ only by 1bp each, and SMA diagnosis might be performed by single-strand conformational polymorphism, PCR amplification followed by restriction fragment length polymorphism (RFLP), multiple ligation–dependent probe amplification, or real- time PCR of SMNs exons 7 and 8. We developed a simpler and cost-effective method to detect SMN1 exon 7 deletion based on allele-specific amplification PCR. Introduction S pinal muscular atrophy (SMA, MIM #253300), the leading genetic cause of death in childhood, is an au- tosomal recessive neuromuscular disorder characterized by progressive muscle weakness resulting from degeneration and loss of the anterior horn cells in the spinal cord and the brain stem nuclei. Although the clinical spectrum indicates a continuous variation, SMA is classically subdivided into three types depending on the age of onset and the severity of symptoms (Munsat and Davies, 1992). SMA is associated to deletions of the survival motor neu- ron (SMN) gene identified and mapped to chromosome 5q13, a complex region at the genomic level, including a 500- kbp duplication and inversion (Lefebvre et al., 1995). SMN is present in two highly homologous copies, both containing nine exons. Most of the chromosomes of normal individuals (noncarriers) have at least one telomeric (SMN1) and one centromeric (SMN2) copy, although 5% of chromosomes with an intact SMN1 gene exist that lack SMN2 (Lefebvre et al., 1995). Approximately 95% of patients suffering from different forms of SMA carry homologous deletions of SMN1 exon(s) 7 (and 8). SMN1 and SMN2 exons 7 and 8 differ only by 1 bp each, which makes the standard deletion analysis by PCR am- plification not feasible, but allowed the development of other molecular methods for SMA diagnosis either based on single-strand conformational polymorphism (SSCP) (Le- febvre et al., 1995) or by PCR amplification using mismatched primers followed by restriction analysis (van der Steege et al., 1995), multiple ligation–dependent probe amplification (MLPA) (Arkblad et al., 2006; Scarciolla et al., 2006), or real-time PCR for SMN copy number (Feldko ¨ tter et al., 2002). We developed a simpler and cost-effective method to detect both SMN1 and=or SMN2 exon 7 deletion, based on allele- specific amplification PCR (PCR-ASO), without the use of a fluorescently labeled primer as previously proposed (Moutou et al., 2001). Patients and Methods Fifty SMA patients were diagnosed according to the con- sensus criteria (Munsat and Davies, 1992) and by means of PCR amplification followed by SSCP analysis (Lefebvre et al., 1995). They were characterized as SMA1 (n ¼ 24), SMA2 (n ¼ 13), and SMA3 (n ¼ 13). The duplex PCR-ASO proposed was developed with a previously described forward primer R111 5 0 -AGACTATCAACTTAATTTCTGATCA-3 0 (Lefebvre et al., 1995; Gavrilov et al., 1998), and two newly designed reverse primers, with the 3 0 -end specific to the C- to T-transition that distinguishes SMN1 and SMN2: 7C1r, 5 0 -CTT CTTTTTGATTTTGTCTG-3 0 specific for SMN1 exon 7; 7T2r, 1 Laborato ´ rio de Pediatria Clı ´nica—Lim 36; 2 Unidade de Neurologia Infantil, Instituto da Crianc ¸a; 3 Divisa ˜o de Clı ´nica Neurolo ´ gica; 4 Unidade de Gene ´tica, Instituto da Crianc ¸a; Hospital das Clı ´nicas, Faculdade de Medicina da Universidade de Sa ˜o Paulo, Sa ˜ o Paulo, Brazil. GENETIC TESTING AND MOLECULAR BIOMARKERS Volume 13, Number 2, 2009 ª Mary Ann Liebert, Inc. Pp. 205–208 DOI: 10.1089=gtmb.2008.0066 205