Allele-specific amplification for preimplantation genetic diagnosis (PGD) of spinal muscular atrophy

• Published in: Prenatal diagnosis 2001-06, Vol.21 (6), p.498-503
• Main Authors: Moutou, Céline, Gardes, Nathalie, Rongières, Catherine, Ohl, Jeanine, Bettahar-Lebugle, Karima, Wittemer, Christiane, Gerlinger, Pierre, Viville, Stéphane
• Format: Article
• Language: English
• Subjects: Adult; allele-specific amplification; Alleles; Biological and medical sciences; Birth control; Blastomeres; DNA Primers; Female; Gene Amplification; Gynecology. Andrology. Obstetrics; Humans; Lymphocytes; Male; Medical sciences; Muscular Atrophy, Spinal - diagnosis; Muscular Atrophy, Spinal - genetics; Mutation - genetics; Nucleic Acid Amplification Techniques; Polymerase Chain Reaction - methods; Pregnancy; Preimplantation Diagnosis - methods; preimplantation genetic diagnosis (PGD); single cell PCR; spinal muscular atrophy; Sterility. Assisted procreation
• ISSN: 0197-3851; 1097-0223
• DOI: 10.1002/pd.110

We have developed a new allele‐specific amplification method for the preimplantation genetic diagnosis (PGD) of spinal muscular atrophy (SMA; Werdnig‐Hoffmann disease) from a single cell. This method is based on the detection of the deletion of exon 7 of the telomeric copy of the survival motor neurone (SMNt) gene. An oligonucleotide was designed to be specific to the SMNt nucleotidic sequence with exonic mismatch G (for SMNt)→A (for SMNc) at its 3′ end. This test produces reliable PCR products in 95% of single lymphoblasts (85/88) tested as well as in 16/16 blastomeres from normal controls. Specificity analysis showed that we were able to detect homozygous deletion of the SMNt gene in 99% of single lymphoblasts (103/104) from a SMA patient. No contamination was detected in 68 blanks tested. Multiple cell and DNA dilution analysis revealed that the test is accurate and specific up to 100 pg DNA and should thus also be suitable for PGD at the blastocyst stage. This rapid procedure requires a single round of fluorescent PCR and no restriction digestion, while previously described single cell methods include nested PCR followed by restriction enzyme digestion. Two PGD cycles for SMA using this procedure were performed in our centre. Copyright © 2001 John Wiley & Sons, Ltd.