Genetic Analysis of Taiwanese Patients with malignant hyperthermia

中文摘要 ( Abstract in Chinese ) 背景： 惡性高溫(MH)為一與骨骼肌病變有關的體染色體顯性遺傳疾病，主要由吸入性麻醉劑和去極化之肌肉放鬆劑所引發的病症。第一型理阿諾鹼受體(RYR1)基因，為細胞內鈣離子釋放通道，被認為是造成惡性高溫的原因之一。到目前為止，至少有30個RYR1突變點和惡性高溫有關。在本研究中我們收集本土在臨床上發生惡性高溫病人，針對RYR1進行DNA突變分析，我們以RYR1突變的熱區(hot spots)作為研究對象，並以DHPLC的方法來篩選其突變點。 方法和結果： 我們收集了本土的惡性高溫病人五名和五十名健康人的血液，抽取血中的去氧核醣核酸（DNA）。我們挑選了RYR1 exon 6、9、11、12、14、15、17、39、40、45和46進行聚合酶連鎖反應(PCR)。將所得聚合酶連鎖反應之產物進行高效能液體層析法(DHPLC)分析，以層析圖找出含有異結合體的樣本後，再以去氧核糖核酸定序法去找出其變異的氮鹼。 在五個惡性高溫病人中，我們發現了四個突變，分別是：Tyr522Cys、Arg552Trp、Val2168Met、Thr2206Arg。其中有兩個病人的突變點相同為Thr2206Arg。在這四個突變中有三個曾被報告過而有一個是嶄新的(Tyr522Cys)。 結論： 我們利用高效能液體層析法去篩選RYR1的突變熱區，我們成它a在這五個病人身上都找出致病突變。在五位惡性高溫病人中我們發現了四個RYR1 DNA突變點，其中Tyr522Cys是新的突變點。DHPLC提升了篩選DNA突變的效率，而國人突變的熱區和西方報告相同。英文摘要( Abstract in English )： Background： Malignant hyperthermia (MH) is an autosomal-dominant inherited disorder of the skeletal muscle that can be triggered by commonly used volatile anesthetic agents and depolarizing muscle relaxants. The ryanodine receptor of skeletal muscle (RYR1), which is an intracellular calcium release channel, has been proposed to be a candidate gene for the MH defect. To date, at least 30 RYR1 mutations have been described. In this study, we enrolled 5 Taiwanese patients with MH and used DHPLC techniques to screen the mutation hot spots of RYR1 gene. Methods and Results： We enrolled 5 Taiwanese patients with MH and 50 healthy volunteers. We set up PCR protocol to amplify RYR1 exon6、9、11、12、14、15、17、39、40、45、46.We used denaturing HPLC (DHPLC) to analysis RYR1 PCR products. Upon detection of a heterozygous elution pattern in DHPLC analysis, we performed DNA sequencing reaction on these samples and identified the variations in nucleotides. We identified four different mutations in the 5 patients with MH. These mutations were Tyr522Cys、Arg552Trp、Val2168Met、Thr2206Arg. Among them, there were 2 patients having the same mutation, Thr2206Arg. Three of the mutations have been reported before and one (Tyr522Cys) was a novel mutation. Conclusion： We used DHPLC to screen the mutation hot spots of RYR1 gene and identified the mutations in 5 Taiwanese patients with MH. We identified four mutations and one of them was novel. DHPLC enhanced the efficacy of mutation screening. We found that RYR1 mutation hot spots in Taiwanese were similar to those in Western people.目錄 ( Contents ) 縮寫表 ( Abbreviation ) 中文摘要 ( Abstract in Chinese ) 1 英文摘要 ( Abstract in English ) 3 緒論 ( Introduction ) 5 材料與方法 ( Materials and Methods ) 21 結果 ( Results ) 28 討論 ( Discussion ) 34 結論與展望 ( Conclusion and Prospective ) 40 參考文獻 ( Reference ) 4