title: Duplex PCR for preimplantation genetic diagnosis (PGD) of spinal muscular atrophy.
abstract: The main difficulty in developing a molecular diagnosis of spinal muscular atrophy (SMA) resides in the specific genomic structure of the locus. Indeed, two highly homologous survival motor neurone genes, SMN1 and SMN2, are present at the locus. The detection of the homozygous deletion of exons 7 and 8 of the SMN1 gene, which is present in 90 to 98% of the patients, is based on methods highlighting 1 of the 8 nucleotidic mismatches existing between these 2 genes. In order to offer preimplantation genetic diagnosis (PGD) for SMA, we developed a new allele-specific amplification method. The main disadvantage of our previously described strategy resided in the possibility of diagnosing, in case of amplification failure, an unaffected embryo as affected. We present here a new PGD-SMA method. We established the conditions for three different duplex PCRs, allowing the specific detection of the SMN1 gene and one polymorphic marker, either D5S629, D5S1977, or D5S641. Of the 60 to 90 single cells tested, the PCR efficiency varied from 98 to 100% with a complete genotype obtained in a range between 81 and 87% with a global allele drop-out rate of 9%. Such a test was used to perform 1 PGD cycle for which 7 embryos could be analysed. All the embryos were fully diagnosed, six as unaffected and one as affected. Four embryos were transferred, but no pregnancy ensued.
authors.affiliation: Service de Biologie de la Reproduction-SIHCUS-CMCO, CHU de Strasbourg, Schiltigheim Cedex, France.
authors.forename: Céline
authors.initials: C
authors.lastname: Moutou
authors.forename: Nathalie
authors.initials: N
authors.lastname: Gardes
authors.forename: Stéphane
authors.initials: S
authors.lastname: Viville
mesh_terms: D000328:Adult; D001706:Biopsy; D017362:Cyclic AMP Response Element-Binding Protein; D004624:Embryo Transfer; D004622:Embryo, Mammalian; D005091:Exons; D005260:Female; D017353:Gene Deletion; D005838:Genotype; D006801:Humans; D008214:Lymphocytes / Q000737:chemistry; D008297:Male; D008875:Middle Aged; D009419:Nerve Tissue Proteins / Q000235:genetics; D010375:Pedigree; D016133:Polymerase Chain Reaction / Q000379:methods*; D019836:Preimplantation Diagnosis / Q000379:methods*; D016601:RNA-Binding Proteins; D055532:SMN Complex Proteins; D014897:Spinal Muscular Atrophies of Childhood / Q000175:diagnosis* / Q000235:genetics*; 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; 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