許多人類因三聯核酸重複序列擴增突變(expansion mutation)所導致的遺傳性神經及肌肉退化性疾病，大部分是由於CAG與其互補序列CTG所引起的。CAG擴增突變通常發生在密碼區(coding region)
，而CTG擴增突變通常發生在不轉譯區(untranslated region)。之前我們將長的CTG與CAG重複序列接在GFP基因的不轉譯區，注射到線蟲(C. elegans.)體內，發現到不論是CTG或CAG的重複序列都會對線蟲造成生理上的影響。
本研究中我們將不同長度CAG repeats接在綠螢光蛋白(EGFP)基因之3′UTR (3’untranslated region)，並以gamma-sarcoglycan (GSG)基因之promoter來驅動綠螢光蛋白在老鼠肌肉專一性表達。結果我們發現(CAG)200轉殖基因鼠的綠螢光蛋白表現量會明顯的降低，而從出生時就已經開始。且(CAG)200轉殖基因鼠初代肌肉細胞核內會有RNA foci的產生，但(CAG)200轉殖基因鼠在表型上卻無肉眼可分辨的明顯異常。在生理及小鼠的行為表現上，我們發現(CAG)200的轉殖基因公鼠較難使母鼠懷孕。在肌肉細胞的部份，會出現非典型的肌肉細胞特徵如：細胞核的數目較多，且有些核會位於細胞的中央。在琥珀醯酸去氫?與NADH還原?的病理染色中，我們發現(CAG)200小鼠肌肉細胞含多較高活性琥珀醯酸去氫?，而NADH還原?的染色紋路也有異常。我們所觀察到的結果可以肯定(CAG)200的基因轉殖小鼠其肌肉細胞已出現明顯的病理變異，但此變異是局部性的。在肌肉電生理方面，我們並沒有很明顯的看見(CAG)200有肌強直的情形。(CAG)200小鼠的成鼠肌肉細胞中MyoD、Myf 5、myogenin、及CHCR等肌肉分化指標基因(muscle differentiaion marker gene)有高量表現。
這些實驗結果，我們首次發現位於3端非轉譯區(3’UTR)的CAG repeat不僅會影響到簡單動物，如: 線蟲的蟲體肌肉活動力下降及神經傳導異常，也會影響到哺乳類動物，如: 小鼠肌肉及細胞型態改變、肌肉細胞分化遲緩及神經傳導受到影響。目前雖然沒有研究報告指出有哪一種人類神經肌肉方面的疾病或是其他方面的疾病是由3’UTR之CAG三聯核酸擴增突變所造成的，然而，這並不意味著沒有此一可能性，只是尚未知道。
Among human trinucleotide repeat disorders, the most frequent triplets found to be expanded are CAG and its complementary sequence, CTG. CAG repeats are almost always found in coding regions whereas CTG expansions are located in untranslated regions (UTRs). Previously we showed that expanded CTG and CAG repeat in the 3’-UTR of GFP both had pathogenic effect in transgenic C. elegans. To investigate the possible pathogenic effect of untranslated (CAG)n trinucleotide repeat in mammals, we made transgenic mice expressing a muscle-specific transcript with (CAG)200 inserted in the 3’-UTR of EGFP gene. The long tract of CAG repeat was found to cause reduced protein expression of EGFP as evidenced by direct fluorescence and western blotting. Histological analysis of the muscle sections revealed atypical muscle cell morphology as well as abnormal staining patterns of succinate dehydrogenase and NADH activity in (CAG)200 mice. Furthermore, mice expressing expanded CAG repeat exhibited a slight deviation in muscle tension recording, suggesting a sign of low muscle activity. By RNA fluorescent in situ hybridization, the muscle cells of (CAG)200 mice were shown to contain nuclear retentions of transcripts, or nuclear foci. Further analysis demonstrated that the expression of muscle differentiation markers, MyoD, Myf5, myogenine and CHCR, were upregulated in the muscle cells of adult (CAG)200 mice. These effects were specific to CAG repeat because mice expressing EGFP RNA without CAG repeat did not differ from the nontransgenic mice in any aspects as mentioned above. This result is the first to show that the expanded CAG repeat in UTR could cause pathogenic effects in mice and suggested that disorders linked to untranslated CAG repeat expansion may exist in human.
