Failure to detect a DM1 expansion using triplet repeat-primed PCR. J.S. Parboosingh, R.J. Klock, P.J. Bridge Dept Medical Genetics, Alberta Children's Hosp, Calgary, AB, Canada.

   Recently, the CTG repeat expansion in the DMPK gene causing myotonic dystrophy failed to be detected in two patients by triplet repeat-primed PCR (tpPCR) using frequently cited primers. In recent years, this method has been applied to a number of trinucleotide repeat disorders as it has several advantages over the traditional Southern blot analysis. Triplet-primed PCR allows for a rapid turnaround time on a small amount of DNA particularly important for diagnostic confirmation in hypotonic babies and prenatal cases; however, it does not allow one to estimate the size.
   TpPCR has been the method of choice in the Molecular Diagnostic lab for 20 months. During this time 40 screens have been performed for a variety of reasons including: confirmation of diagnosis, and prenatal testing. A two step approach is taken: 1) primers flanking the repeat are used to determine the number of repeats within the normal range and up to approximately 100 repeats; and 2) tpPCR is performed on all samples with a single repeat from step 1 to exclude the presence of a large expansion and thus confirm homozygosity. We have designed tpPCR assays utilizing both DNA strands (using both a CTG and CAG repeat primer) allowing for bidirectional tpPCR. We have detected 17 patients with expansions. Two of the 17 expansions were not detectable using the frequently cited P1 primer with the CTG repeat primer but were detectable using the complementary CAG repeat primer and the opposite flanking primer (other strand). Utilization of tpPCR in only one direction would have led to a false negative result for these patients. This has led to the identification in the SNP database of a C>T substitution within the P1 primer binding site. We will present these findings as well as a frequency for this polymorphism.