title: Efficient and highly sensitive screen for myotonic dystrophy type 1 using a one-step triplet-primed PCR and melting curve assay.
abstract: Instability and expansion of the DMPK CTG repeat cause myotonic dystrophy type 1 (DM1), the most common adult-onset neuromuscular disorder. Overlapping clinical features between DM1 and other myotonic disorders necessitate molecular confirmation for definitive diagnosis. Preconception screening could improve reproductive planning especially in DM1-affected women, who show diminished ovarian reserve and unfavorable in vitro fertilization-preimplantation genetic diagnosis outcome. We optimized triplet-primed PCR and melting curve analysis on 17 DNAs from DM1-affected/unaffected cell lines. A blinded test was performed on 60 genotype-known clinical samples. Plasmid constructs pDMPK(CTG)35 and pDMPK(CTG)48 were used to establish threshold temperatures separating DM1-affected from unaffected samples. Postscreen triplet-primed PCR amplicon sizing was achieved by short-cycle labeled-primer extension followed by capillary electrophoresis. Triplet-primed PCR melting curve analysis melt peak temperatures of unaffected and DM1-affected samples were lower and higher than the control plasmids' melt peak temperatures, respectively. Capillary electrophoresis of post-melting curve analysis amplicons was completely concordant with the screening results. Triplet-primed PCR melting curve analysis is a simple and cost-effective screening tool for rapid identification of DM1. The companion confirmation protocol allows quick determination of CTG repeat size when required. This strategy avoids the need to perform capillary electrophoresis sizing on all test samples, limiting capillary electrophoresis analysis to only a subset of cases that are screen-positive.
authors: Department of Pediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
authors: Mulias
authors: M
authors: Lian
authors: Department of Pediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
authors: Indhu-Shree
authors: IS
authors: Rajan-Babu
authors: Department of Pediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
authors: Kunal
authors: K
authors: Singh
authors: Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore; Division of Medical Sciences, National Cancer Center, Singapore; Duke-NUS Graduate Medical School, Singapore.
authors: Caroline G
authors: CG
authors: Lee
authors: Department of Pediatric Medicine, KK Women's and Children's Hospital, Singapore.
authors: Hai-Yang
authors: HY
authors: Law
authors: Department of Pediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore; Khoo Teck Puat - National University Children's Medical Institute, National University Health System, Singapore; Department of Laboratory Medicine, National University Hospital, Singapore. Electronic address: paecs@nus.edu.sg.
authors: Samuel S
authors: SS
authors: Chong
mesh_terms: D002460:Cell Line; D019075:Electrophoresis, Capillary; D006801:Humans; D009223:Myotonic Dystrophy / Q000175:diagnosis* / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D013696:Temperature