Genetic Analysis of Osteogenesis Imperfecta in Taiwan

摘 要 先天性成骨不全症（Osteogenesis imperfecta；O.I.），是一種因為骨骼結締組織異常所造成的遺傳疾病，其遺傳模式有自體顯性及自體隱性，發生率約為十萬分之6-7，目前臨床依照傳統的分型可以分成四型，在臨床診斷主要依照臨床表徵，然而我們的研究將進行相關基因檢測以及在基因型與表現型相關之探討。 我們進行了100個家族之先天性成骨不全症相關基因檢測，利用PCR、HRM、DHPLC以及基因定序等分生技術來完成，首先針對自體顯性的COL1A1、COL1A2基因來做檢測，在檢測未發現突變且並沒有家族史之家族，再繼續做自體隱性的CRTAP、LEPRE1和PPIB基因之檢測。 台灣在先天性成骨不全症之分佈O.I. type I為28%（26/92）、O.I. type II為12%（11/92）、O.I. type III為36%（33/92）、O.I. type IV為24%（22/92）。在這100個家族的基因檢測結果，46個家族有COL1A1基因的突變、25個家族有COL1A2基因的突變，在COL1A1、COL1A2基因的檢測率為71%，我們在從COL1A1、COL1A2基因檢測為陰性的家族，在PPIB基因之檢測發現到有1個家族有PPIB基因的突變（1/19）。 我們建立台灣在先天性成骨不全症之基因分析流程及資料庫，可以了解到台灣族群基因突變之點位以及易突變區域之分佈，進而快速完成基因檢測，提供在遺傳諮詢時明確的基因變異報告，以及在產前診斷相關之應用，在未來可做為先天性成骨不全症之基因及治療等研究之依據。鍵詞：先天性成骨不全症，基因檢測，COL1A1基因，COL1A2基因，RTAP基因，LEPRE1基因，PPIB基因Abstract Osteogenesis imperfecta (OI) is a heritable disorder of bone formation﹐which is characterized by bone fragility and low bone mass﹒As reported, Autosomal dominant OI is associated with the mutations of COL1A1 and COL1A2 genes. Autosomal recessive OI is association of CRTAP﹑LEPRE1 and PPIB gene﹒ In this study, exon-wide mutation analysis of COL1A1﹑COL1A2﹑CRTAP﹑LEPRE1and PPIB genes in osteogenesis imperfecta originated from Taiwan population by molecular diagnosis coupling several mutation detection techniques including Denaturing High Performance Liquid Chromatography (DHPLC), High-Resolution Melting analysis (HRM), and automated sequencing analysis was performed. One hundred unrelated patients and their family members diagnosed with OI clinically were enrolled in this study. Forty-six patients had mutation in COL1A1 gene﹐25 in COL1A2 gene. The mutation detection rate of COL1A1 and COL1A2 genes is 71%﹒Furthermore, 19 patients without mutation identified in COL1A1/ COL1A2 gene were analyzed for the possible mutations in CRTAP﹑LEPRE1and PPIB genes﹒One patient had mutation in PPIB gene was identified (1/19). No mutation in CRTAP or LEPRE1 gene was found. To gain more insight into the mutational spectrum in Taiwanese patients with OI﹐ we conducted this study to perform extensive exon-wide mutational analysis of COL1A1﹑COL1A2﹑CRTAP﹑LEPRE1and PPIB genes based on the high- throughput mutation scanning system with DHPLC and HRM system﹒There are several hot mutation regions in COL1A1 and COL1A2 gene had been proposed﹒However, further studies to elucidate the genotype-phenotype correlation are still warrant.ey word：Osteogenesis imperfecta﹐mutation analysis﹐COL1A1 gene﹐COL1A2 gene﹐CRTAP gene，LEPRE1 gene﹐PPIB gene﹐High-Resolution Melting analysis (HRM)﹐Denaturing High Performance Liquid Chromatography (DHPLC)目 錄目錄..................................................i目錄..................................................ii文摘要................................................iii文摘要................................................iv一章 緒論...........................................1.1 骨骼之結構....................................1.2 膠原蛋白之結構................................2.3 COL1A1和COL1A2基因之介紹......................3.4 膠原蛋白之顯性負向效應........................3.5 LEPRE1、CRTAP、PPIB基因之介紹.................4.6 臨床表現與診斷................................5.7 疾病分型......................................5.8 先天性成骨不全症之發生率......................6.9 治療..........................................6.10 遺傳諮詢......................................6.11 研究動機......................................7二章 實驗材料與儀器.................................8.1 實驗材料.......................................8.1.1 先天性成骨不全症基因檢測之DNA檢體.................8.1.2 引子對............................................8.1.3 聚合酶連鎖反應試劑................................8.1.4 洋菜膠電泳試劑....................................8.1.5 DHPLC試劑組......................................9.1.6 HRM試劑組........................................9.1.7 DNA定序試劑......................................9.2 實驗儀器.......................................9.2.1 磁珠分離式核酸萃取系統...........................9.2.2 PCR熱循環機......................................9.2.3 DHPLC分析儀......................................9.2.4 HRM分析儀........................................9.2.5 DNA序列分析儀....................................9三章 實驗方法......................................10.1 DNA抽取.......................................10.2 聚合酶連鎖反應（PCR）.........................10.3 洋菜膠電泳（Agarose gel electrophoresis)......10 3.4 DNA突變分析（DHPLC）..........................10.5 高解析基因突變篩檢分析（HRM）.................11 .6 正常族群單一核苷酸多型性分析..................11.7 自動定序分析（Automated sequencing）..........11四章 實驗結果......................................12.1 先天性成骨不全症之疾病表現型與臨床表徵之統計..12.2 先天性成骨不全症之基因檢測結果................12.3 先天性成骨不全症疾病的表現型與基因型之相關....13.4 COL1A1、COL1A2基因與突變形態之分佈統計........14.5 第一型前膠原蛋白α1和α2功能區上表現型與突變 形態之分佈統計................................15.6 COL1A1、COL1A2基因高發生突變區域..............15.7 先天性成骨不全症相關基因之單一核苷酸多型性....16五章 討論..........................................17六章 結論..........................................23七章 參考文獻......................................2