title: Multiplex nested PCR for preimplantation genetic diagnosis of spinal muscular atrophy.
abstract: OBJECTIVE
  Spinal muscular atrophy (SMA) is a common autosomal recessive neuromuscular disorder caused in most patients by homozygous deletion of the SMN1 gene. For a carrier couple at a 25% risk of affected offspring, preimplantation genetic diagnosis (PGD) offers an alternative to prenatal diagnosis and termination of affected pregnancies. Our objective was to develop an accurate and reliable single-cell multiplex nested PCR analysis for PGD of SMA.
  
  
  METHODS
  The method was developed on single blood leukocytes, obtained from healthy controls and an adult SMA type III patient with a known homozygous deletion of SMN1 exon 7 and 8. Multiplex nested PCR on single cells was used to co-amplify exons 7 and 8 of SMN. Additional multiplexing was performed with the ZFX/ZFY gene for sexing. Following successful establishment of the multiplex nested PCR protocol in single leukocytes, the technique was employed for PGD in 4 patients for a total of 7 cycles. In 2 patients, sexing was simultaneously performed using ZFX/ZFY.
  
  
  RESULTS
  220 single leukocytes from a normal individual and 220 from an SMA patient were analyzed. Exon 7 of SMN1 was amplified in 99% of normal single leukocytes and in none of the SMA-affected leukocytes. Exon 7 of SMN2 was amplified in 100% of both normal and SMA-affected leukocytes. Exon 8 of SMN1 was amplified in 98% of normal cells and in none of the SMA-affected leukocytes. Exon 8 of SMN2 was amplified in 96% of both normal and SMA-affected leukocytes. Amplification efficiency was 99% for ZFX/ZFY. There were no false-negative results and no contamination was detected in all wash-drop blanks tested. Seven PGD cycles were performed in 4 SMA-carrier couples with successful molecular analysis of 34 embryos and a total of 15 normal embryos transferred in 7 cycles. One clinical pregnancy has resulted in the delivery of a healthy male. Amniocentesis performed at 17 weeks confirmed the correct diagnosis for both SMA and sexing.
  
  
  CONCLUSIONS
  These results suggest that our multiplex nested PCR protocol offers an efficient and accurate method for PGD of SMA while enabling the simultaneous analysis of an additional loci.
authors.affiliation: Sara Racine in vitro Fertilization Unit, Lis Maternity Hospital, Tel Aviv, Israel.
authors.forename: Mira
authors.initials: M
authors.lastname: Malcov
authors.forename: Tamar
authors.initials: T
authors.lastname: Schwartz
authors.forename: Nava
authors.initials: N
authors.lastname: Mei-Raz
authors.forename: Dalit Ben
authors.initials: DB
authors.lastname: Yosef
authors.forename: Ami
authors.initials: A
authors.lastname: Amit
authors.forename: Joseph B
authors.initials: JB
authors.lastname: Lessing
authors.forename: Ruth
authors.initials: R
authors.lastname: Shomrat
authors.forename: Avi
authors.initials: A
authors.lastname: Orr-Urtreger
authors.forename: Yuval
authors.initials: Y
authors.lastname: Yaron
mesh_terms: D000328:Adult; D020732:Cytogenetic Analysis / Q000379:methods*; D005260:Female; D006801:Humans; D008297:Male; D009134:Muscular Atrophy, Spinal / Q000175:diagnosis* / Q000235:genetics*; D009865:Oocytes / Q000502:physiology; D016133:Polymerase Chain Reaction / Q000379:methods*; D011247:Pregnancy; D019836:Preimplantation Diagnosis / Q000379:methods*