謝豐舟臺灣大學：分子醫學研究所曾淑真Tseng, Su-ChengSu-ChengTseng2007-11-262018-07-092007-11-262018-07-092005http://ntur.lib.ntu.edu.tw//handle/246246/51390A型及B型血友病是因為分別缺乏凝血因子VIII和IX所造成的,為最普遍的遺傳性出血性疾病,在人類為性隱性遺傳。其突變型式範圍很廣,包括染色體本身的反轉突變,例如凝血因子VIII的intron 1 和 22,另外還有許多不同型式的突變,包括刪除、插入和點突變所造成的nonsense、 missense和 splicing 位置的突變。血友病病人本身及家屬所遭遇的問題包含了疾病所帶來的痛苦及死亡離別的問題,社會經濟及負擔的問題,防止疾病的再現,帶因者的基因檢測和產前診斷是很重要的。在此篇研究論文中我們結合了次循環長片段聚合酶連鎖反應及變性高液相色層分析法來篩檢大部份的凝血因子VIII和IX的突變。在44個台灣血友病家族中, 29個為A型血友病, 15個為B型血友病,在研究中我們找到了10個突變位置是之前未被發表過的,8個在A型血友病,2個在B型血友病,另外有13個為凝血因子VIII的Intron 22 反轉突變,其餘的突變點之前已描述過。這些突變都經過更進一步的直接定序確認,也沒發現偽陽性或偽陰性。結合次循環長片段聚合酶連鎖分析和變性高效液相色層分析法分析血友病帶因者的基因及產前診斷,是正確性高且快速同時花費較其他傳統方法低的有效方法。Hemophilia A (HA) and hemophilia B (HB), with the deficiency of coagulation factor VIII (FVIII) and IX (FIX) respectively, represent the most common sex-linked inherited bleeding disorders in human. A wide range of different mutations have been identified including the intrachromosomal inversions involving regions in intron 1 and 22 of the FVIII gene as well as many mutation types found in the remaining part of the factor gene, sunch as large and small deletions, insertions, and point mutations. Patients suffering from those disorders and their families bear great financial and social burden, it is very important to prevent recurrence of the diseases. To achieve this goal genetic analysis for carrier screening and prenatal diagnosis is mandatory. We have established a diagnostic strategy consisting of screening for most common mutations in the Factors VIII and Factor IX genes by using long-distance polymerase chain reaction (LD-PCR)and denaturing high performance liquid chromatography (DHPLC). Forty-four affected Taiwanese families including 29 HA and 15 HB families were analyzed. We found eight novel point mutations in 6 HA families and 2 HB families together with two novel small deletion mutations in HA family.The intron 22 inverions in 13 HA families and the remaining mutations have been described previously in HA and HB mutation database. These small mutations were further confirmed by direct sequencing. Neither false positive nor false negative results were found. Our combinatory approach by subcycling LD-PCR and heteroduplex analysis based on DHPLC proves to be a highly informative, rapid and practical means to detect mutations in affected individuals and carriers of hemophilias.簡稱或縮寫對照表…………………………………………………4 中英文摘要………………………………………………………..6 一、 前言…………………………………………………………8 二、 緒論…………………………………………………………11 血友病的歷史沿革…………………………………………12 臨床表現……………………………………………………13 遺傳表現……………………………………………………14 篩檢試驗……………………………………………………14 分子遺傳試驗………………………………………………14 產前檢查……………………………………………………15 遺傳諮詢…………………………………………………..15 處理…………………………………………………………16 三、 研究動機……………………………………………………17 四、 實驗材料與儀器……………………………………………18 五、 實驗方法及步驟……………………………………………20 六、 結果…………………………………………………………23 七、 討論…………………………………………………………25 附録一. DHPLC的分析原理………………………………………….31 附錄二. 次循環長片段聚合酶連鎖反應之原理…………………34 附錄三. A型血友病國外報導突變型式所占的比例………………35 附錄四. B型血友病國外報導突變型式所占的比例……………36 附錄五. 凝血因子VIII的結構圖………………………………37 圖表……………………………………………………………….38 參考資料………………………………………………………….632042911 bytesapplication/pdfen-US長片段聚合酶連鎖反應變性高液相色層分析血友病台灣人DHPLCTaiwanesehemophiliaLD-PCR[SDGs]SDG3otherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/51390/1/ntu-94-P92448001-1.pdf