Pathogenic role of ribonuclear inclusions in myotonic dystrophy type 2: myth or reality?

Objectives Myotonic dystrophy type 2 (DM2) is caused by a CCTG expansion in intron 1 of the zinc finger protein 9 gene which generates a toxic RNA implicated in the pathology of the disease. Mutant transcripts are retained in muscle nuclei producing ribonuclear inclusions (RIs), which result in misregulated splicing events that explain many symptoms of DM2, such as myotonia and insulin resistance. Nevertheless the cause of skeletal muscle degeneration, which is a cardinal feature of the disease, remains still unknown. In order to study if RIs and the subsequent nuclear sequestration of MBNL1, a splicing factor, are involved in DM2 muscle wasting, we have correlated the degree of fibres atrophy with the number of RIs or with the dimension of RIs and MBNL1 nuclear foci. Methods The study has been carried out on 4 DM2 patients who underwent 2 different biopsies of biceps brachii at different years of age. Fibre diameters have been evaluated on cryostat sections immunostained with fast or slow myosin. Moreover, we have performed FISH in combination with fast myosin-immunofluorescence to evaluate the number of RIs present in nuclei of the two fibre types, or FISH in combination with MBNL1-immunofluorescence to quantify fluorescence intensity and area of RIs and MBNL1 foci. Results A preferential type 2 fibre atrophy has been observed in both the biopsies examined from each DM2 patients. The number of RIs appears to be similar in fast and slow fibres indicating that no correlation exists between the degree of fibre atrophy and the number of RIs. A slight increase in fluorescence intensity and area of both RIs and MBNL1 foci is observable in the biopsies of each patient examined. Indeed it has been demonstrated that in DM2 patients expansion size increase with patient’s age. Nevertheless this increase is not always accompanied by an increase of the number of RIs or of the degree of fast fibre atrophy. Conclusions Our data indicate that higher fast fibre atrophy is not accompanied to a higher number of RIs in this type of fibres as compared to slow fibres. The increase in fluorescence intensity and area of both RIs and MBNL1 foci in the second biopsy seems indicate an increase in expansion size and in MBNL1 sequestration over the time. However this increase is not related to a worsening of histopathological pattern. The number and the dimensions of ribonuclar inclusions and MBNL1 do not seem to be indicators of the severity of muscle wasting.