非轉譯區內CTG三聯核酸重複序列之擴增突變已知會造成人體最常見之遺傳性神經肌肉方面的疾病，稱為強直型肌肉萎縮症第一型(Dystrophia myotonia type 1, DM1)。 由於此一疾病之病徵多出現於青春期之後，在此研究中我們利用線蟲想了解擴增之CTG及CAG三聯核酸重複序列致病效應與個體發育之關係。首先，我們將帶有不同序列長度之綠螢光蛋白(GFP)基因接入含線蟲肌肉細胞專一性驅動的載體。經顯微注射產生之線蟲共有myo-3::gfp，myo-3::gfp(CAG)5，myo-3::gfp(CTG)5，myo-3::gfp(CAG)120，及myo-3::gfp(CTG)120五種。經螢光顯微鏡分析結果顯示擴增之CTG及CAG序列皆使得GFP表現量明顯降低，而此螢光亮度之減弱開始於L3階段，至Adult階段幾乎已無法偵測。觀察線蟲各個時期的爬行軌跡顯示myo-3::gfp(CAG)120及myo-3::gfp(CTG)120線蟲在L3時期開始出現較清楚異常軌跡，顯示肌肉活動之不協調性。成蟲時，其爬行速度亦大幅下降。利用phalloidin- rhodamine染色分析發現L3至L4時期其異常型態之肌肉細胞數目顯著增加。電子顯微鏡觀察清楚顯示myo-3::gfp(CAG)120蟲體肌肉細胞間之dense body已不存在，而在myo-3::gfp(CTG)120蟲體，原本存在肌肉束與表皮細胞之間的basement membrane，亦被發現在肌肉細胞內的肌肉束中間。進一步以螢光原位雜交技術我們發現GFP-(CUG)120 foci僅存在於成蟲細胞核內，L2幼蟲中並不存在，顯示RNA foci之形成亦與發育有關，並可能因而造成GFP表現量的減少以及引發病理機制。這些實驗結果首次證明過長CAG重複序列亦會造成線蟲肌肉結構功能喪失，而無論是擴增CTG或CAG序列之影響皆與發育有密切關係。
Expansion of CTG trinucleotide repeats located in the 3’ untranslated region (UTR) of myotonin protein kinase (DMPK) gene causes the most common adulthood neuromuscular disease, myotonic dystrophy 1 (DM1). Most classical DM1 patients did not start showing clinical phenotypes until young adulthood. To provide a model system for investigating the developmentally regulated pathogenic effects of DM1, we have examined the phenotypes of Caenorhabditis elegans expressing green fluorescent protein (GFP) gene with 0, 5, or 120 CTG and CAG repeats in the 3’-untranslated region driven by the myo3 promoter. Transgenic worms expressing GFP-CUG120 and GFP-CAG120 RNA, but not those expressing GFP-CUG5 and GFP-CAG5RNA, displayed an age-dependent impairment of GFP expression, locomotory behavior, moving rate, and muscle structure. In addition, RNA foci were found in the nuclei of the adult, but not L2, myo3::GFP-CTG120 animals, suggesting that the RNA foci formation may correlate with the decrease of GFP expression and the muscular dysfunction. These results first demonstrate that both the expanded CUG and CAG repeats could cause developmentally regulated defects on gene expression and muscle structure in C. elegans.
