Assessment of Preimplantation Genetic Diagnosis (PGD) for Childhood-onset Spinal Muscular Atrophy (SMA) Using Duplex Fluorescent PCR

Document Type: Research Paper


1 Department of Genetics and Molecular Biology, Isfahan University of Medical Sciences, P.O. Box: 81744-176, Isfahan, Iran.

2 Department of Anatomical Sciences, School of Medicine, Isfahan University of Medical Sciences, P.O. Box: 81744-176, Isfahan, Iran.


Preimplantation genetic diagnosis (PGD) is a very early form of prenatal diagnosis (PND) by which genetic
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.