Usefulness of fluorescent repeat-primed PCR for the detection of complex variants of CTG repeats associated with myotonic dystrophy type 1 (DM1) not revealed by routine diagnostic PCR techniques

To attest the higher sensitivity of repeat primed polymerase chain reaction (PCR) versus routine diagnostic PCR protocols to amplify complex variant repeats at the 3'-end of the CTG array associated with Myotonic Dystrophy Type 1 (DM1).
We performed a clinical-molecular characterization in two unrelated patients manifesting a Steinert' disease phenotype, yet resulted negative for DM1 testing at two different certified Genetic Laboratories. Our protocol of study included molecular testing for DM1 and DM2 by routine PCR based techniques: in particular DM1 molecular testing consisted of a two- step PCR procedure (a first step of PCR reaction able to detected either wild-type and small-expanded alleles followed, in the case of amplification of only one allele, by a second step of long PCR /Southern blotting protocol to detect expanded alleles). We also performed muscle biopsy studies, including RNA fluorescence in situ hybridization (RNA-FISH) using either CAG and CAGG labeled probes, as well as an analysis by RT-PCR of the expression genes known to be aberrantly spliced in DM1 tissues. Finally, a fluorescent repeat-primed PCR protocol was applied to definitively assess the presence of CTG expansions at the DM1 locus.

Both patients manifested myotonia, cranial, bulbar, axial and semi-distal muscle weakness and atrophy, cataract, cardiac and endocrine disturbances. Molecular testing for DM2 was negative while, according to the results of routine DM1 testing, both patients resulted homozygous for a wild-type allele. Muscle morphology showed increased central nuclei, fiber size variability and predominant type 1 atrophy. RT-PCR studies documented an aberrant alternative splicing of several genes usually affected in myotonic dystrophy muscle tissues. Interestingly RNA-FISH on muscle biopsies documented in both cases the presence of CUG-repeat positive RNA foci . Therefore we decided to further assess the presence of CTG expansions at the DM1 locus. Finally we were able to amplify the expanded CTG repeat at the DM1 locus in both patients using a fluorescent repeat-primed PCR protocol.
Fluorescent repeat-primed PCR confirms to be a sensitive, easy and time consuming procedure in order to detect complex variants of CTG repeats, estimated to be present in about 3-4% of DM1 patients and usually not revealed by routine diagnostic PCR techniques. Therefore we propose to include this technique protocol among the PCR-based protocols widely applied for DM1 molecular testing.

No relevant conflicts of interest declared.