In all DM2 patients,

a single PCR product representing the normal allele can be identified because the DNA polymerase fail to amplify the mutant allele due to length and #selleckchem randurls[1|1|,|CHEM1|]# stable secondary structure. All individuals showing two alleles for the marker are excluded from having the DM2 mutation. However, identical allele size on two normal alleles occurs in 12% of the population; (B) all patients appearing to have one allele Inhibitors,research,lifescience,medical need further molecular analysis to determine whether or not they carry a DM2 expansion. Because of the incomplete sensitivity of Southern analysis, a DM2 repeat assay (RP-PCR) was developed; (C) the RP-PCR method involves amplifying the CCTG repeat by PCR, and probing the resultant product with an internal probe to assure specificity. The combined use of

these methods allows 99% sensitivity and specificity for known expansions. Several alternative and highly sensitive methods have been developed for DM2 mutation verification including long-range PCR (80) and a tetraplet-primed PCR (81). A Inhibitors,research,lifescience,medical modified Southern method using field–inversion electrophoresis (FIGE) is particularly efficient in determining the mutation length (82). However, these methods are still too long and complicated to be part of routine laboratory diagnostics. Nevertheless ribonuclear foci and splicing changes are present before any histological abnormality manifestations (43, 83). This could be important for an early diagnosis Inhibitors,research,lifescience,medical before the spectrum of clinical signs of muscle disease appear. So a more practical tool to obtain a definitive DM2 diagnosis in few hours is represented by in situ hybridization (ISH) which is a method that allows the direct

sellekchem visualization of the mutant RNA on muscle biopsy (84, 85). By using specific probes Inhibitors,research,lifescience,medical for CCUG expansions, it permits a differential diagnosis between DM2 and DM1. Therefore it may be a simple approach for DM2 diagnosis, which can be performed in a rapid and sensitive manner in any pathology Inhibitors,research,lifescience,medical laboratory. ISH with CAGG probe should be considered as a routine laboratory procedure to confirm or refute the clinical suspicion of DM2. It should also be applied routinely to screen patients Brefeldin_A with myotonic disorders (84, 85). This approach makes muscle biopsy an essential tool for DM2 diagnosis (Fig. 1A). Moreover, since MBNL1 is sequestered by mutant RNA foci, it is possible to visualize the nuclear accumulation of MBNL1 by immunofluorescence on muscle sections (Fig. 1B). However, although MBNL1 represents an histopathological marker of DM, it does not allow to distinguish between DM1 and DM2 (86). Figure 1. Fluorescence in situ hybridization (FISH) in combination with MBNL1-immunofluorescence on DM2 muscle section. A. Visualization of (CCTG)n expansion on muscle section by FISH using (CAGG)5 specific probe. Red spots within myonuclei (blue, DAPI) represent … Muscle biopsy The histological features of muscle in DM1 and DM2 are very similar (Fig.