Juvenile case of myotonic dystrophy type 2 correlates with early spliceopathy : biomolecular evidence for anticipation?

Objective: Myotonic dystrophy type 2 (DM2) is a common adult onset muscular dystrophy caused by a dominantly transmitted CCTG expansion in intron 1 of ZNF9 gene. In DM2 there is no obvious evidence for an intergenerational increase of expansion size and no congenital cases have been reported. We describe clinical, histopathological, genetic and molecular analysis in a 14-year-old female with juvenile onset DM2 and her affected mother presenting with a more severe phenotype and a later onset of symptoms. Methods: Biceps brachii muscle biopsy and blood samples were taken from two DM2 patients. Muscle tissue was fresh-frozen in isopentane cooled in liquid nitrogen for routine histological or histochemical stainings, fast and slow myosin isoforms and CLC1 immunohistochemical stainings and FISH in combination with MBNL1-immunofluorescence. Genetic characterization of the DM2 mutation has been obtained by a combination of long-PCR and Southern blot analyses on DNA extracted either from peripheral blood and muscle. The RT-PCR splicing assays for the IR, MBNL1 and CLCN1 genes have been carried out on muscle samples. Results: Histological and immunohistochemical analysis do not correlate with disease severity or age at onset in both patients. Only a small increase in the CCTG repeat number through maternal transmission. FISH in combination with MBNL1-immunofluorescence on muscle sections shows the presence of mutant-mRNA and MBNL1 nuclear foci whose fluorescence intensity and area appear to be similar in the two patients. Splicing analysis of the IR, ClCN1 and MBNL1 genes in muscle tissue demonstrates that the level of aberrant splicing isoforms is lower in the daughter than in the mother. No mutations of the CLCN1 gene have been found excluding a second genetic mutation in this ion channel gene responsible for myotonia in the young DM2 patient. Conclusion: Juvenile onset with early abnormal expression of splicing products should be also considered in DM2. These findings might be the basis for biomolecular anticipation in DM2. Alternative mechanisms should be to show clinical and biomolecular differences in comparison to DM1