建立基因突變診斷技術與基因劑量分析系統於基因相關疾病之研究

Abstract

自從邁入二十一世紀以來，隨著基因體計畫逐步的進行，有愈來愈多的疾病被證實與基因突變有關。而這些疾病，又會隨著地域之分布有所不同。但是，絕大部分之單一基因疾病，一方面由於檢驗技術耗時繁瑣，常需要量身訂做，無法大量制式化施行檢驗。而另一方面，這些疾病極為罕見，醫療費用又高昂，在現行健保制度下常常成為孤兒，在各大醫學中心並非發展重點。但是，提供這些單一基因疾病正確之基因診斷，不論是對於病患本身治療，或是給予遺傳諮詢，以及進一步實施下一代產前基因診斷種種方面，都是極為重要的。 未來的生物醫學，尤其在臨床基因醫學方面，將有革命性的單一核苷酸多型性﹙single nucleotide polymorphism, SNP﹚之應用。愈來愈多的單一核苷酸變異已被確認，並且開啟了一道提供我們發現更多致病基因、藥物動力學，及了解人類起源之署光。以往的生物醫學分析系統，在使用上不但耗時、耗人力，並且藥劑的使用量也相當的大，造成檢驗成本的大幅提高。基於上述理由，快速基因診斷技術之發展日漸蓬勃，因此希望發展一套快速、有效率、可靠且不昂貴之偵測系統來運用於基因定量與基因診斷平台，以快速檢驗方式，可以在短時間內有效率、經濟及準確的篩檢出重症患者及帶因者，以便日後臨床檢驗的大量使用。 本論文利用 DNA 片段突變分析儀﹙Denaturing High Performance Liquid Chromatography, DHPLC﹚，自動化之偵測來找出單一核苷酸之變異。利用 DNA 片段突變分析儀之特性，完成多項之基因檢測項目，包括甲/乙型海洋性貧血、結節性硬化症、杜顯氏/貝克氏肌肉萎縮症、脊髓性肌肉萎縮症等等，提供於臨床診斷及基礎研究所需。另外，利用引子延長法﹙primer extention﹚之技術，再結合 DHPLC 之高效率與快速分析平台，發展新一代快速基因檢測平台，可以達到單一核苷酸變異檢測之目標。 另外，目前發現有愈來愈多之疾病和基因數量上之改變有關聯。其中最廣為人知的就是屬於大段基因之缺失或是大段基因異常複製。對於基因數量上之改變，基因定量系統之建立顯得困難許多。基於現今常用以PCR為基本技術之突變偵測模式，常常是只能用於定性而非專長於定量。 在本論文中建立了一套快速，有效率、可靠且較不昂貴之基因定量分析方法於人類不同種類基因疾病。運用DNA突變分析儀與毛細管電泳﹙Capillary electrophoresis﹚，此兩種技術平台來建立基因定量快速分析系統。於此論文之中，我們針對不同基因之特性設計不同之定量方式及多重引子對之偵測策略，而將其運用於包括杜顯氏/貝克氏肌肉萎縮症、脊髓性肌肉萎縮症、小胖威利症、巧口-瑪利-吐司氏病及甲型地中海貧血之基因檢測之上。除此之外，一旦我們建立了此套新式快速基因定量檢測模式，我們將可更進一步將其運用於許多臨床上三倍數染色體疾病，如唐氏症、Trisomy 13，Trisomy 18，或是一些常見之基因大段缺失之症候群的快速診斷，如DiGeorge，William症候群等等。 此外，聚丙氨酸異常擴增相關疾病目前已被發現為一群全新類型的重覆核苷酸異常擴增疾病。這一類疾病和之前所知的重覆核苷酸異常擴增疾病有許多之不同。首先，帶有聚丙氨酸擴增特徵之基因多是和發育相關之重要轉錄因子。所以，臨床上這類基因突變所造成之臨床症狀常和先天畸型有關。目前之證據顯示，聚丙氨酸異常擴增相關疾病之病理機轉乃由於蛋白質的異常折疊及裂解所造成，所以，其臨床之致病機轉應也是與參與此過程之因子有關。 為了釐清及進一步研究聚丙氨酸異常擴增之分子機轉及功能，本論文利用PHOX2B基因為標的，建立一套臨床快速及可靠之基因分析系統。在此章節中，利用毛細管電泳發展精確、快速、再現性高的分析技術。此技術將可供臨床診斷及後續研究，更進而以利其他聚丙氨酸異常擴增基因診斷之所需。 為了更進一步準確的提高偵測率及診斷效率，此論文更發展出許多快速基因診斷平台。本論文嘗試利用melting curve analysis及MLPA技術建立可靠、快速又經濟的分子診斷方法，。利用高速、經濟而有效率之基因診斷工具，在基因突變診斷與基因劑量分析中變得極為重要。醫學上利用DHPLC、CE、melting curve analysis、MLPA之技術來從事基因診斷，其特點為DHPLC具強大之隨機突變點搜尋能力，而毛細管電泳可以成為基因劑量分析之平台，melting curve analysis提供了相當便宜且有效率的篩檢平台，MLPA可偵測全基因的基因劑量。和傳統之突變分析方法比較，臨床上利用DHPLC、CE、melting curve analysis及MLPA之技術，將可使基因診斷變得更有效率敏感且更符合經濟規模效益。

As we are now entering the post-genomic era, there are a number of new research trends. One trend will interrogate the influence of common genetic variants within the human genome. These common variants, which are often referred as polymorphisms, have already been proven to play a role in genomic disease, drug toxicity, and general pharmaceutical efficacy. Genetic polymorphisms (particularly single-nucleotide polymorphisms, SNPs, which account for ~90% of common genetic variants) are currently used within various association studies for disease research. Another trend will be an increase in the number of disease is that the various genetic mutations within a functional pathway and reveal their linkages to the diseases. This genetic various on the functional regions within the genome, including trinucleotide repeat expansions, associated with genetic disorders caused by misfolded protein. The third trend is to provide clinical genetic diagnosis by utilizing the available SNP and mutation analytical tool for earlier disease prevention, prenatal diagnosis and health care. In this application, accuracy, speed, automation, reliability, affordability, and flexibility, as well as the ability to detect both known and unknown mutations will be of great importance. These three trends alone demonstrate the post-genomic era’s requirements for genetic analysis technologies that can provide high degrees of sample throughput without sacrificing sensitivity, as well as high levels of automation without sacrificing flexibility. To address these needs, we present a new nucleic acid analysis technology, including denaturing high-performance liquid chromatography (DHPLC), capillary electrophoresis (CE), melting curve analysis and MLPA. The DHPLC have proven to be a promising tool for nucleic acids separation. The success of the DHPLC approach to genetic analyses is demonstrated in its applications by genetic mutation and polymorphism discovery and screening in many commercial clinical diseases diagnosis. We develop two DHPLC methods including heteroduplex analysis and single base primer extension, to optimize the efficiency in genetic diagnosis for analysis of genes with unknown mutations, genes with hundreds of mutations but no hot spots and genes with hot-spot mutations. In this dissertation, we established the efficient and accurate DHPLC platform provide available testing procedures and protocols applying on several entities of diseases, such as alpha-thalassemia, beta-thalassemia, tuberous sclerosis complex, Duchenne/ Becker muscular dystrophy, and spinal muscular atrophy. Although many genetic diseases are caused by the presence of point mutations in respective genes, an increasing number of diseases are known to be caused by gene copy number changes. We want to develop a rapid and reliable method for quantitative analyses for human genome in various genetic disorders. The method involves amplifications of a test locus with unknown copy number and a reference locus with known copy number, following by DHPLC and CE techniques to detect single copy changes without the use of radioactive labeling. In this dissertation, we established the efficient and accurate gene dose determination system by using the DHPLC and CE platform based on multiplex PCR strategies and applying on several entities of diseases including Duchenne/ Becker muscular dystrophy, spinal muscular atrophy, Prader-willi syndrome, Charcot-Marie-Tooth disease and alpha-thalassemia. Moreover, once we have established this powerful system, we will further apply this technique on rapid detection of trisomy syndrome and microdeletion syndrome including trisomy 13, trisomy 18, DiGeorge syndrome, William’s syndrome, and others. Moreover, Polyalanine repeat expansions constitute a new group of repeat expansion disorders that differ from previously known expansion-associated diseases in many ways. They occur primarily in transcription factors with important roles during development. As a consequence, the clinical spectrum associated with these mutations consists mainly of congenital malformation syndromes. Based on the finding that protein misfolding and degradation are a major pathogenetic mechanism in polyalanine repeats, it is likely that the clinical phenotype is influenced by the expression of chaperones and other factors that affect this process. To explore the genetic regulating mechanism of polyalanine expansion and the pathophysiologic pathway, we conduct this pilot study. In the dissertation, we established the reliable comprehensive genetic testing for PHOX2B gene and for the other genes with abnormal polyalanine expansion. In summary, by using DHPLC, CE, melting curve analysis and MLPA techniques in genetic diagnosis, the advantages including that DHPLC got the powerful ability to identify random mutations, CE can the be adapted to gene dosage determination for high throughput screening in medical applications, melting curve analysis could identify the mutation site with highly efficient performance and MLPA allows detection of gene deletions, duplications, and rearrangements in whole gene. Compared to classic approaches of mutation screening, DHPLC, CE, melting curve analysis and MLPA could be valuable alternative in a more rapid, economic and highly sensitive way in genetic diagnosis.