Margolis Jamie M, Schoser Benedikt G, Moseley Melinda L, Day John W, Ranum Laura P W
Department of Genetics, Cell Biology and Development, University of Minnesota, 420 Delaware Street SE, Minneapolis, MN 55455, USA.
Hum Mol Genet. 2006 Jun 1;15(11):1808-15. doi: 10.1093/hmg/ddl103. Epub 2006 Apr 19.
Myotonic dystrophy type 2 (DM2) is caused by a CCTG expansion mutation in intron 1 of the zinc finger protein 9 (ZNF9) gene. The mean expansion size in patients is larger than for DM1 or any previously reported disorder (mean=5000 CCTGs; range=75-11 000), and similar to DM1, repeats containing ribonuclear inclusions accumulate in affected DM2 tissue. Although an RNA gain-of-function mechanism involving DM1 CUG or DM2 CCUG expansion transcripts is now well established, still debated are the potential role that flanking sequences within the DMPK 3'-UTR may have on disease pathogenesis and whether or not decreased expression of DMPK, ZNF9 or neighboring genes at these loci contribute to disease. To address these questions in DM2, we have examined the nucleic acid content of the ribonuclear inclusions and the effects of these large expansions on ZNF9 expression. Using cell lines either haploid or homozygous for the expansion, as well as skeletal muscle biopsy tissue, we demonstrate that pre-mRNAs containing large CCUG expansions are normally spliced and exported from the nucleus, that the expansions do not decrease ZNF9 expression at the mRNA or protein level, and that the ribonuclear inclusions are enriched for the CCUG expansion, but not intronic flanking sequences. These data suggest that the downstream molecular effects of the DM2 mutation are triggered by the accumulation of CCUG repeat tract alone.
2型强直性肌营养不良症（DM2）由锌指蛋白9（ZNF9）基因内含子1中的CCTG重复扩增突变引起。患者中该重复序列的平均扩增长度大于1型强直性肌营养不良症（DM1）或之前报道的任何疾病（平均=5000个CCTG；范围=75 - 11000），并且与DM1类似，含有核糖核蛋白包涵体的重复序列在受影响的DM2组织中积累。尽管涉及DM1的CUG或DM2的CCUG扩增转录本的RNA功能获得机制现已明确，但仍存在争议的是，DMPK 3'-UTR内的侧翼序列在疾病发病机制中可能发挥的潜在作用，以及这些位点上DMPK、ZNF9或邻近基因的表达降低是否会导致疾病。为了解决DM2中的这些问题，我们检测了核糖核蛋白包涵体的核酸含量以及这些大的重复扩增对ZNF9表达的影响。使用对于该重复扩增为单倍体或纯合子的细胞系以及骨骼肌活检组织，我们证明含有大量CCUG重复扩增的前体mRNA能够正常剪接并从细胞核输出，这些重复扩增在mRNA或蛋白质水平上不会降低ZNF9的表达，并且核糖核蛋白包涵体富含CCUG重复序列，但不包含内含子侧翼序列。这些数据表明，DM2突变的下游分子效应仅由CCUG重复序列的积累引发。
