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

Document Type: Research Paper

Authors

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.

Abstract

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.

Keywords