Mutant (CCTG)n Expansion Causes Abnormal Expression of Zinc Finger Protein 9 (ZNF9) in Myotonic Dystrophy Type 2

Abstract

The mutation that underlies myotonic dystrophy type 2 (DM2) is a (CCTG)n expansion in intron 1 of zinc finger protein 9 (ZNF9). It has been suggested that ZNF9 is of no consequence for disease pathogenesis. We determined the expression levels of ZNF9 during muscle cell differentiation and in DM2 muscle by microarray profiling, real-time RT-PCR, splice variant analysis, immunofluorescence, and Western blotting. Our results show that in differentiating myoblasts, ZNF9 protein was localized primarily to the nucleus, whereas in mature muscle fibers, it was cytoplasmic and organized in sarcomeric striations at the Z-disk. In patients with DM2 , ZNF9 was abnormally expressed. First, there was an overall reduction in both the mRNA and protein levels. Second, the subcellular localization of the ZNF9 protein was somewhat less cytoplasmic and more membrane-bound. Third, our splice variant analysis revealed retention of intron 3 in an aberrant isoform, and fourth quantitative allelespecific expression analysis showed the persistence of intron 1 sequences from the abnormal allele, further suggesting that the mutant allele is incompletely spliced. Thus, the decrease in total expression appears to be due to impaired splicing of the mutant transcript. Our data indicate that ZNF9 expression in DM2 patients is altered at multiple levels. Although toxic RNA effects likely explain overlapping phenotypic manifestations between DM1 and DM2, abnormal ZNF9 levels in DM2 may account for the differences in DM1.

RNA Foci, CUGBP1, and ZNF9 Are the Primary Targets of the Mutant CUG and CCUG Repeats Expanded in Myotonic Dystrophies Type 1 and Type 2

“…Reduction of ZNF9 in DM2 might be also associated with the accumulation of soluble CCUG repeats or nonspliced ZNF9 pre-mRNA. 29,31 Because the pathology of DM1 and DM2 is caused by long CUG and CCUG repeats correspondingly, the general assumption is that the nondegraded CUG/CCUG repeats are toxic. However, recent data suggest that high levels of short repeats might also be toxic.…”

“…One of the critical alterations in DM2 muscle is the reduction of protein levels of ZNF9. 29,31,48 However, the role of CCUG repeats in the reduction of ZNF9 levels requires additional investigation as some patients with DM2 show reduction of ZNF9, but others do not. 29,31,34,48,49 The analysis of CUGBP1 levels in DM2 also provides controversial results, showing an increase of CUGBP1 in muscle biopsies from some DM2 patients and no changes of CUGBP1 in other patients with DM2.…”

“…29,31,48 However, the role of CCUG repeats in the reduction of ZNF9 levels requires additional investigation as some patients with DM2 show reduction of ZNF9, but others do not. 29,31,34,48,49 The analysis of CUGBP1 levels in DM2 also provides controversial results, showing an increase of CUGBP1 in muscle biopsies from some DM2 patients and no changes of CUGBP1 in other patients with DM2. 31,35,48 To examine whether low levels of CCUG repeats target ZNF9 and CUGBP1, we analyzed the effect of a single pulse of transcription of short and long CCUG repeats on ZNF9 and CUGBP1 levels.…”

Cardiac Involvement in Myotonic Dystrophy Type 2 Patients With Preserved Ejection Fraction

“…In DM2, the zinc finger protein is genetically changed, 5 leading to systemic disease with alteration of several organs.…”

Myotone Dystrophien: Klinik, Pathogenese, Diagnostik und Therapie

“…CNBP (ZNF9) ist ein RNA und DNA bindendes Protein[68], das wahrscheinlich die Funktion eines Translationsfaktors (reguliert die Translation) ausübt[69,70]. Auch wenn toxische RNA-Effekte die phänotypische Variabilität der DM2 erklären, trägt wahrscheinlich auch die verminderte Expression von CNBP zum Phä-notyp bei[71]. Unterstützt wird diese Hypothese durch die Tatsache, dass ZNF9-mRNA und ZNF9-Protein bei DM2 vermindert sind, dass das ZNF9-Protein nicht im Zytoplasma, sondern membrangebunden vorkommt und dass die mutierten Sequenzen im Intron 1 persistieren[71].…”