Upgrading molecular diagnostics of myotonic dystrophies: multiplexing for simultaneous characterization of the DMPK and ZNF9 repeat motifs

Myotonic dystrophy (DM) is a common neuromuscular disorder comprising at least two genetically different forms. DM1 is caused by expansion of a (CTG)(n) repeat in the DMPK gene, while DM2 is caused by expansion of a (CCTG)(n) part of a complex repetitive motif (TG)(n)(TCTG)(n)(CCTG)(n) in the ZNF9 gene. Detection of the responsible expansions is complicated in both cases because of the extremely variable length of the expanded alleles, which can contain even several thousands of repeats in both disorders. One of the commonly used detection approaches utilizes the combination of conventional PCR and "triplet" or "tetraplet" repeat-primed PCR (TP-PCR). TP-PCR can be performed simultaneously or successively in both DM1 and DM2 testing. We have designed two multiplex reactions which include bi-directionally labelled conventional PCRs and TP-PCRs for both DM1 and DM2 loci. These two reactions can be used under the same amplification and electrophoretic conditions thus allowing their parallelisation into a one step method. Simultaneous analysis of the samples using these two multiplex reactions allows characterization of both the DM1 and DM2 repeat regions in the time usually required for the first screening step in conventional DM1 or DM2 testing.