Assessment of preimplantation genetic diagnosis (PGD) for childhood-onset spinal muscular atrophy (SMA) using duplex fluorescent PCR

Preimplantation genetic diagnosis (PGD) is a very early form of prenatal diagnosis (PND) by which gene-tic diagnosis is performed on a single embryonic blastomer obtained by embryonic biopsy. Hence the genetic status of the embryo can be defined prior to embryo transfer; thus termination of pregnancy would not happen. The objective of the present paper was to establish preimplantation diagnosis for spinal muscular atrophy (SMA), a prevalent monogenic disorder in Iran, and to find the efficacy and sensitivity of the developed protocol. The fluorescent duplex single cell PCR technique for detection of SMN gene exon 7 deletion was developed first on single lymphocyte and then on a single blastomer. The protocol was duplexed with one of the three linked polymorphic markers namely, D5S112, D5S435 and D5S679. The PCR products of SMN exon 7 were restricted with DraI restriction endonuclease. Linkage analyses through polymorphic markers were also used for diagnosis. The amplification rates for SMN exon 7 deletion on a single lymphocytes and single blastomers were 96 and 88% respectively, using the aforementioned methods. The mean amplification rates of 3 polymorphic markers were 94% on single lymphocytes and 84% on single blastomers, respectively. Mean allele drop out (ADO) for the 3 markers on single lymphocytes was 4%. In conclusion the developed duplex single cell fluorescent PCR seems to be an accurate and feasible method at single cell level and could be easily applied in clinical preimplantation genetic diagnosis.