Incidence of amplification failure in DMPK allele due to allelic dropout event in a diagnostic laboratory

Background: Myotonic dystrophy type 1 (DM1) is caused by an expanded CTG repeat in the non-coding 3’ UTR of the DMPK gene. PCR and Southern Blot Analysis (SBA) of long-range PCR represent the routine molecular testing most widely used for DM1 diagnosis. However, in these conventional methods artifacts such as allele dropout (ADO) represent a risk of misdiagnosis for DM1. Subjects, who show a single product by conventional methods, require a complementary technique such as triplet repeat primed PCR (TP-PCR). Object: To estimate and minimize the incidence of allele dropout event in our diagnostic molecular laboratory by the use of new kit TP-PCR-based. Methods: We retrospectively studied 190 DMPK alleles, on blood samples from to ninety-five subjects, previously genotyped by traditional methods to validate a new assay. The pedigree of a DM1 family was used to expand our analysis. Results: TP-PCR assay correctly identified all 95/95 (100%) subjects and these results were in agreement with the other molecular laboratory. By conventional methods the amplification failure due to allele dropout in our cohort was in 12/190 (6.3%) DMPK alleles analyzed. When these 12 alleles were detected and solved by new assay, we found that the 2.6% was caused by primer sequence-dependent and the remaining 3.6% by polymerase-hindering secondary structures. Conclusions: Allele dropout could be considered as a potentially important problem in DM1 diagnosis that may lead to the attribution of a wrong genotype with long-term consequences for both proband and family.