ClC1 chloride channel in myotonic dystrophy type 2 and ClC1 splicing in vitro.

Myotonic dystrophy type 2 (DM2) is caused by CCTG-repeat expansions. Occurrence of splicing and mutations in the muscle chloride channel gene CLCN1 have been reported to contribute to the phenotype. To examine the effect of CLCN1 in DM2 in Germany, we determined the frequency of a representative ClC1 mutation, R894X, and its effect on DM2 clinical features. Then, we examined CLCN1 mRNA splice variants in patient muscle functionally expressed the most abundant variant, and determined its subcellular localization. Finally, we established a cellular system for studying mouse clcn1 pre-mRNA splicing and tested effects of expression of (CCUG)₁₈, (CUG)₂₄ and (AAG)₂₄ RNAs. The R894X mutation was present in 7.7% of DM2 families. DM2 R894X-carriers had more myotonia and myalgia than non-carriers. The most abundant CLCN1 splice variant in DM2 (80% of all transcripts) excluded exons 6-7 and lead to a truncated ClC1(236X) protein. Heterologous ClC1(236X) expression did not yield functional channels. Co-expression with ClC1 did not show a dominant negative effect, but a slightly suppressive effect. In C₂C₁₂ cells, the clc1 splice variants generated by (CCUG)₁₈-RNA resembled those in DM2 muscle and differed from those generated by (CUG)₂₄ and (AAG)₂₄. We conclude that ClC1 mutations exert gene dose effects and enhance myotonia and pain in DM2 in Germany. Additionally, the ClC1(236X) splice variant may contribute to myotonia in DM2. Since splice variants depend on the types of repeats expressed in the cellular C₂C₁₂ model, similar cell models of other tissues may be useful for studying repeatdependent pathogenetic mechanisms more easily than in transgenic animals.