Modification of the triplet repeat primed polymerase chain reaction method for detection of the CTG repeat expansion in myotonic dystrophy type 1: application in preimplantation genetic diagnosis

Objective: To overcome problems associated with the use of triplet repeat primed polymerase chain reaction (TP-PCR) in preimplantation genetic diagnosis (PGD) of myotonic dystrophy type 1 (DM1). Design: Clinical research study. Setting: UCL Centre for PGD and Centre for Reproductive and Genetic Health. Patient(s): Seven couples undergoing PGD for DM1. Intervention(s): A modified TP-PCR protocol (mTP-PCR) for the reliable detection of both expanded and nonexpanded alleles in DMPK was optimized using single lymphocytes. Four cycles of PGD were performed with TP-PCR for diagnosis and a further 10 cycles with mTP-PCR. Main Outcome Measure(s): Amplification efficiency, allele dropout, diagnosis rate, and delivery rate. Result(s): Preliminary testing showed that the TP-PCR amplification efficiency was higher using lymphocytes versus buccal cells. Single lymphocytes gave very high amplification efficiencies for both protocols (99% to 100%). There were no false-positive or false-negative results for 148 single lymphocytes tested with mTP-PCR compared with 9% (5 out of 54) false-positive results with TP-PCR, indicating the improved accuracy of the modified protocol. In embryos, the diagnosis rate was 95.6% with mTP-PCR and 75% with TP-PCR. Conclusion(s): For PGD of DM1, mTP-PCR is recommended. It may also be applied as a rapid screen for DMPK expansions in individuals with symptoms of DM1, relatives of known mutation carriers, or in prenatal diagnosis. (Fertil Steril (R) 2010;94:1674-9. (C) 2010 by American Society for Reproductive Medicine.)