| Abstract: | 肌強直肌肉萎縮症(myotonic dystrophy, dystrophia myotonia, DM)是位於第十九對染色體上之DMPK (myotonic dystrophy protein kinase)基因第15個exon上一段CTG三聯核酸重複序列發生擴增突變(expansion mutation)所造成，而CTG的擴增長度和DM臨床症狀的嚴重性以及患者發病年齡有很大的關係。傳統上CTG擴增突變之偵測以南方點漬法(Southern blot)為主，PCR技術之發展使得DM分子診斷更為簡單方便，然而PCR的應用上易受限於不良品質的模版及高比例(C+G)的CTG重複區域，為了改進這些缺點，我們發展了FTAR-PCR分析系統。此系統不僅操作方便而且非常的靈敏，它包括了檢體的收集、保存、DNA純化及PCR分析序程，是一套非常完整的偵測系統。利用此系統只需以微量的DNA或極少數細胞，即可偵測出CTG重複序列數目。此外我們以口腔內膜細胞以及羊水細胞作為檢體，開發此分析系統在DM盛行率調查及產前診斷方面的應用，研究結果顯示FTAR-PCR系統不僅可應用於DM遺傳諮詢及臨床診斷上，未來應可以用於其他三聯核酸增生疾病的偵測。
先前研究發現具有不同CTG序列長度之alleles 與存在CTG重複序列上游5 kb左右之Alu insertion/deletion多型性有聯鎖不平衡之關係，並據以推測CTG擴增突變途徑係經由(CTG)5-10，跳過(CTG)11-13，到(CTG)19-30，而最後至(CTG)>50。透過研究DM及正常alleles與Alu多型性之關係，我們發現CTG之擴增突變途徑可能並非如Imbert等人提出之跳躍式突變，而是連續式突變，也就是說，CTG序列突變過程是隨機、漸進式的由5次增加至50次以上。煞而經調查CTG序列長度與CTG區域附近多個單一鹼基多型性之聯鎖關係，結果顯示台灣與歐美地區DM alleles有相同之單套型，符合DM來自於一個或少數祖先之說法(founder effect)。而初步單套型分析結果亦符合連續式突變之假說。未來，透過更完整之檢體收集及單套型分析將能進一步闡釋CTG之擴增突變途徑。
Myotonic dystrophy (DM) is caused by a CTG trinucleotide expansion mutation at exon 15 of the myotonic dystrophy protein kinase (DMPK) gene. The clinical severity of this disease correlates with the length of the CTG trinucleotide repeats as well as the age of onset. Determination of the CTG repeat length has been primarily relied on Southern blot analysis on restriction enzyme-digested genomic DNA. The development of PCR technology provides a much more sensitive and simpler protocol for DM diagnosis. However, the quality of the template and the high (G+C) ratio of the amplified region hamper the use of PCR on the diagnosis of DM. Here we describe a procedure, FTAR-PCR system, including sample collection, DNA purification, and PCR analysis of CTG repeat length without using 7-deaza-dGTP. This protocol is very sensitive and convenient since high quality DNA sample can be obtained with only few nucleate cells, including lymphoblastoid cells, buccal cells, and amniotic fluid cells, for the detection of CTG repeat length. Therefore, FTAR-PCR system could be very useful in genetic counseling and prenatal diagnosis as well as prevalence study of other trinucleotide repeat diseases.
By studying the linkage disequilibrium between Alu polymorphism and various CTG repeat lengths， Imbert et al. proposed that the DM mutations were derived from the pool of alleles with 19-30 CTG repeats, and themselves derived from a very small number of ancient expansions from the major (CTG)5 allele. However, our data indicated that the expansion pathway could be continuous, that is, the mutation process is random, progressive from (CTG)5 to (CTG)50. Through the study of the relationship between CTG repeat lengths and several single base polymorphism present flanking the CTG region, our results showed that the DM alleles in Taiwanese population have a specific haplotype, which is the same as that observed in Caucasian population. Since our preliminary result is consistent with the continuous model of CTG expansion, a more complete haplotype analysis including additional markers using (CTG)18-40 alleles will further elucidate the CTG mutation pathway. |
