使用變性高效能液相層析法（DHPLC）來建立高效率的台灣人馬凡氏症基因突變分析方法以及資料庫

Abstract

馬凡氏症是一種自體顯性遺傳疾病，是由於連接組織出問題所導致，其中主要可分為三大類：骨骼，眼睛，以及心臟血液系統等。依國外統計數據，其發生率約每五千人就會有一人是馬凡氏症的患者（1/5,000 ~ 1/20,000），目前已知大多數的患者是由於FBN1基因（Fibrillin gene）突變所導致，此基因位於染色體15q21.1的位子，於1991年被定位出來；而少數的患者則是由於TGFBR2基因（Transforming growth factor-β 2 receptor gene）突變所導致，此基因是於2004年才被定位出來的，位於染色體3p24.1的位子，命名為馬凡氏症第二型基因。 本論文所提出的方法是利用聚合酶連鎖反應（PCR）加上DNA片段突變分析儀（DHPLC）來建立快速且可信的台灣人馬凡氏症的檢測平台同時建立台灣人的馬凡氏症突變點位（mutation site）和多型性點位(polymorphism site)的資料庫。在此研究中，將DNA的來源分成兩部分，一部分是有家族史的個案，共有22個家族，另一部分是偶發性的個案，共有43個個案；結果顯示，若DNA片段突變分析儀（DHPLC）有偵測到的個案，再使用定序（Sequence）方法確認，皆可得到同樣的結果；對於點突變的檢測而言，有家族史的檢測率是77.3%，沒有家族史的檢測率是32.6%。 相較於傳統的基因突變檢測方法，例如：Single Strand Conformation polymorphism（SSCP）或是直接定序（Sequence），在此篇論文當中所提及到的結合聚合酶連鎖反應（PCR）加上DNA片段突變分析儀（DHPLC）不僅可提供一種高效率、高準確率、高可信度、又可節省金費的技術平台，並可同時建立台灣人馬凡氏症的基因資料庫，由於此基因相當大，共有65 exons，若有資料庫可供人參考，便可將所節省的時間用於其他更需要的地方。

Marfan syndrome (MFS) is an autosomal dominant disorder of connective tissue, and its cardinal manifestations involve the skeletal, ocular, and cardiovascular systems. According to the statistical series, its prevalence is between 1/5,000 ~ 1/20,000. We now know that MFS was caused by mutations of FBN1 （Fibrillin gene） at 15q21.1 was reported in 1991. Less frequently, MFS was caused by mutations of TGFBR2 （Transforming growth factor-β 2 receptor gene）at 3p24.1 was reported in 2004. This is named Marfan syndrome type II. In the thesis, we established a rapid and reliable detection system of genetic diagnosis and we constructed database including mutation site and polymorphism site for the Taiwanese patient with Marfan syndrome by using PCR/DHPLC assay. In this study, we decided the original DNA into two groups; one group had family history, including 22 families, and the other group did not have family history, including 43 cases. The result revealed that the data of DHPLC who compatible with the data of sequence, and the mutation detection rate of the cases with family history was 77.3%, and the mutation detection rate of the cases without family history was 32.6%. Comparing with the traditional technology of genetic diagnosis, for example, single strand conformation polymorphism（SSCP）or direct sequence, we demonstrated that the PCR/DHPLC assay was not only an efficient, accurate, reliable, and saving-money technique for the gene diagnosis but also set up the Taiwanese database of the Marfan syndrome to help people who work for the gene testing of FBN1 to saving money and valuable time .