不同基因座內(CTG/CAG)n重複序列長度頻率分佈調查及(CTG/CAG)n基因轉殖線蟲疾病模式建立

之前的研究顯示，部份原發型無精子症(idiopathic azoospermia)患者在X染色體上的男性素接受器(androgen receptor, AR)基因上的一段CAG三聯核酸重複序列有擴增的現象。為了要了解在原發型無精子症患者其他(CTG/CAG)n三聯核酸重複序列的基因座是否也有重複數目之擴增，我們收集了48個原發型無精子症患者與47個正常成年男子的DNA檢體，以聚合酉每聯鎖反應(polymerase chain reaction, PCR)的方法來分析並比較這二類檢體DNA在myotonic dystrophy type 1 (DM1)、Machado-Joseph disease (MJD)、dentatorubral- pallidoluysian atrophy (DRPLA)，和spinocerebellar ataxia type 8 (SCA8)等四個基因座內(CTG/CAG)n三聯核酸重複序列之頻率分佈。雖然這些基因座內(CTG/CAG)n三聯核酸重複數目都在正常範圍，我們發現原發型無精子症的病人較一般人在DM1及MJD基因座內的(CTG/CAG)n三聯核酸重複序列數目明顯偏高，但在DRPLA及SCA8基因座內的(CTG/CAG)n三聯核酸重複序列數目則無顯著差異。此結果顯示，至少在部分原發型無精子症患者，某些疾病基因座內的(CTG/CAG)n三聯核酸重複序列變得較不安定。而是否會因此使得其後代得到三聯核酸重複序列擴增疾病的潛在風險提高，則有待進一步評估。 第一型強直型肌肉萎縮症(myotonic dystrophy type 1; DM1)是一種多系統病變的體染色體顯性遺傳疾病。此疾病的發病原因係因DM1PK (myotonic dystrophy type 1 protein kinase) 基因3’端不轉譯區的一段(CTG)n三聯核酸重複序列發生擴增突變所致。為了瞭解DM1病人體內其他含有(CTG/CAG)n三聯核酸重複序列的基因座是否也具有重複次數增加的現象，我們分析比較了50名DM1患者與80名正常人，在Machado-Joseph disease (MJD)、dentatorubral-pallidoluysian atrophy (DRPLA)，和spinocerebellar ataxia type 8 (SCA8)等三個基因座的(CAG/CTG)n 三聯核酸重複序列重複次數之頻率分佈。所有的檢體都沒有足以致病的三聯核酸重複序列長度。然而，DM1患者在SCA8及MJD基因座中的三聯核酸重複序列重複次數之平均值及分佈趨勢皆明顯地較一般人高(p<0.05)。而DRPLA基因座中兩群體的三聯核酸重複序列重複次數分佈卻沒有明顯的差異。這個結果顯示DM1患者基因體內的(CAG/CTG)n 三聯核酸重複序列在某些基因座的確有較不安定的傾向。 myotonic dystrophy type 1 (DM1)是一種神經肌肉方面的疾病，它是由第19對體染色體上myotonic dystrophy type 1 protein kinase (DM1PK)基因3’端不轉譯區的(CTG)n三聯核酸重複序列擴增突變所引起的遺傳性疾病。在本研究中我們以線蟲作為DM1的疾病模式動物，調查(CTG/CAG)n三聯核酸重複序列對基因表達以及肌肉活動力的影響。我們以具肌肉組織專一性表達之驅動子驅動GFP基因的表現作為報導訊號，在GFP基因的3’端不轉譯區插入重複次數為5、120、和200的(CTG/CAG)n三聯核酸重複序列。我們發現轉殖入(CTG)200的線蟲無法產出F2 line。而轉殖入(CTG)5與(CTG)120的線蟲其GFP蛋白質表達量明顯地比控制組的線蟲低了許多。雖然(CTG)5的線蟲對GFP基因表達的影響與轉殖入(CTG)120的線蟲相去不遠，但我們發現轉殖入(CTG)120的線蟲在活動力的表現上比控制組及轉殖入(CTG)5的線蟲有明顯下降。此外，我們驚訝地發現轉殖入(CAG)5及(CAG)120的線蟲出現與(CTG)5及(CTG)120的轉殖線蟲相同的情況。雖然作用機制仍不清楚，這些結果顯示，3’UTR的(CTG/CAG)n三聯核酸重複序列擴增後的確會抑制其相鄰的基因表達，並造成線蟲肌肉細胞功能之改變。

An increase in CAG trinucleotide repeats length in the androgen-receptor gene has been linked to idiopathic azoospermia. To test whether other (CTG/CAG)n loci are affected in the genomes of azoospermia patients, we investigated the (CAG/CTG)n frequency distribution at myotonic dystrophy type 1 (DM1), Machado-Joseph disease (MJD), dentatorubral-pallidoluysian atrophy (DRPLA), and spinocerebellar ataxia type 8 (SCA8) loci, in 48 azoospermia patients and 47 control individuals. No disease-associated alleles of above loci were found in all samples. However, significant differences were noted in the upper tails of both DM1 and MJD allele distributions. At the DM1 locus, alleles of more than 18 repeats were observed only in azoospermia patients but not in controls (P=0.014). At the MJD locus, the frequency of larger alleles ((CAG)29-37) was also much higher in azoospermia patients (29.2% versus 7.4%; P=0.0001). The DRPLA and SCA8 distribution did not differ in the two populations. These data indicated that at least in a subset of azoospermia patients, there was an increase in the number of trinucleotide repeats in some disease loci. It is therefore noteworthy that a potential risk of increased prevalence of trinucleotide repeats disease may exist in the future generations of these azoospermia patients Myotonic dystrophy type 1 (DM1) is characterized as an autosomal dominant, multisystemic disorder that is caused by expansions of an unstable CTG trinucleotide repeat at the DM1 locus. To investigate whether other (CTG/CAG)n loci are affected in the genomes of DM1 patients, we analyzed the (CTG/CAG)n repeat sizes at Machado-Joseph disease (MJD), dentatorubral-pallidoluysian atrophy (DRPLA), and spinocerebellar ataxia type 8 (SCA8) loci, in 50 DM1 patients and 80 control individuals. No disease-associated alleles of above loci were found in all samples. However, at the MJD and SCA8 loci, the frequency of larger size alleles (>28 repeats) was much higher in DM1 patients than in controls (P=3 x 10-7 and p=2 x 10-4, respectively). Furthermore, the average repeat lengths at these two loci were also significantly longer in DM1 patients (P<0.05). On the other hand, the differences of both the frequency of large size alleles (>20 repeats) and the average repeat lengths at the DRPLA locus in the two studied populations were insignificant (P>0.05). These results first demonstrate that the frequency distributions of large trinucleotide repeats at certain trinucleotide expansion disease loci may also be elevated in DM1 patients. Myotonic dystrophy type 1 (DM1) is a neuromuscular disorder caused by a CTG trinucleotide expansion in the 3’-UTR of the DM1PK gene. In this study we attempt to establish a DM1-disease animal model using transgenic C. elegans. To investigate the effect of (CTG/CAG)n triplets on the gene expression and muscle function, we have made transgenic constructs in which (CTG/CAG)n was inserted into the 3’-untranslated region of GFP gene driven by muscle-specific promoter of C. elegans. Microinjection of (CTG)200 or (CAG)200 constructs failed to produce any line, suggesting that the expression product from these constructs might cause severe defects of muscle function. Four lines, including (CTG)5, (CAG)5, (CTG)120, and (CAG)120, were generated. However, the GFP protein levels in these lines were much lower than that in the GFP-only control lines. By comparing the patterns of body movement, we observed that the (CTG/CAG)120 lines have a significantly lower muscle activity than of (CTG/CAG)5 lines. Although the underlying mechanism remains elusive, these observations demonstrate that the (CTG/CAG)n tract in 3’-UTR could decrease the gene expression in cis, and result in abnormal neuromuscular behavior in vivo.