易脆X染色體症候群分子生物學之研究

第一章易脆X染色體症候群點突變之偵測中文摘要: 易脆X染色體症候群 為常見遺傳性智能障礙疾病﹐與此症候群有關的基因﹐fragile X retardation-1(FMR-1)已被找到﹐導致fragile X syndrome 主要由於 FMR-1基因5端不轉譯區(5UTR untrans)atedregion)之CGG trinucleotide tandem repeat [p(CGG)n]發生倍增突變(expansion mutation)所致﹐其 它原因尚有基因缺失(deletion)及點突變(point mutation)﹐而使FMR-1 基因無法正常表現蛋白或蛋白失去正常功能。 過去我們從406位啟智教 養院之院生中找到29位易脆X染色體症候群可疑患者﹐其FMR-1基因 之( CGG)n三聯核甘酸無倍增現象﹐欲進一步了解FMR-1基因是否發生突變﹐故 以SSCP(singlestrand conformation polymorphism)分析這些人FMR-1基 因第一至第十exon及第十五exon﹐並嘗試找出點突變之位置﹐很幸運發現 五位病人在exon 10區域有異常的SSCP電泳型態其中三位經DNA定序分析後 發現在intron 10的第十四個核甘酸從C變為T。進一步以RT-PCR分析其 mRNA的表現﹐發現exon 10被切掉導致密碼於exon 11發生frameshift造成 提早終止密碼﹐而產生不完全蛋 白產物所致。此外發現其中一位病人除 了有exon 10之point mutation﹐又同時於exon 15也有異常SSCP型態﹐經 DNA定序發現於RGG box中第二個RGG由於核甘酸從G變為A使得原本氨基酸 由Arginine變為Histidine﹐此一突變可能影響蛋白的構型以及與RNA結合 之能力。 本研究主要運用SSCP分析找到FMR-1基因常見點突變之位置﹐ 並希望藉由點突變病人之發現進一步探討FMR-1基因功能與結構之關係。 英文摘要：　　　 In the majority of fragile X syndrome patients, the molecular mechanism of this　disorder is based on an expansion in the 5 untranslated region of the FMR-1 genewhich causes hypermethylation of the CpG island in the promoter region, and subsequently transcriptional suppression of FMR-1 gene.Deletions in the FMR-1 gene have also been shown to cause fragile X syndrome. As to point mutation within the FMR-1 gene, resulting in the same of disorder, there is only two reports beside this study. In this study, we analyzed the FMR-1 gene from 29 patients who have fragile X syndrome phenotype but no (CGG)n trinucleotide expansion in FMR-1. From SSCP analysisof FMR-1 exon 1—10 and 15, we identified two different intragenic mutation in FMR-1: a single base substitution C→T at the 14th nucleotide in the intron 10 causing frameshift on exon 11 and thus,produce pre mature protein; and an missense mutation at exon 15 resulting in an Arg →His substitution. Our results suggest that point mutation in FMR-1 could be responsible for the fragile X syndrome.第二章反轉錄聚合連鎖反應於易脆X染色體症候 群篩檢以及產前診斷之運用中文摘要: 易脆X染色體症候群為常見之遺 傳障礙疾病﹐其發生率僅次於唐氏症﹐導致易脆X染色體症候群之主要原 因係由於FMR-1基因5端不轉譯區CGG三核甘酸重覆序列擴增使得靠近動區 啟CpG island甲基化而造成FMR-1基因不表現所致。傳統以細胞遺傳的方 法誘發X染色體fragile site 來診斷此疾病﹐需培養大量細胞不僅耗時且 並非所有病人皆有fragile site 的產生﹐無法作正確診斷。而南方點墨 式 分析的方法雖能正確的診斷但較耗時﹐且實驗困難度較高。為了開發 一套快速、經濟、準確率高的方法﹐我們嘗試利用RT-PCR的方法針對3位 疑似易脆X染色體症候群病人進行篩檢﹐從中發現三位病人無法偵測到 FMR-1基因表現﹐南方點墨式分析已證實係為FMR-1基因發生突變所致。在 產前診斷應用上﹐目前已完成的個案顯示此方法可檢測出男性胎兒體內 FMR-1基因的正常表現。故運用RT-PCR的分析方法已經可以成功的篩檢到 易脆X染色體的男性病人﹐期待經由不斷的努力改良能使得RT-PCR進入臨 床診斷階段。英文摘要: The fragile X syndrome is the most common genetic cause of mental retardation after Down syndrome. In the vast majority of cases, this is caused by the expansion of an unstable CGG trinucleotide repeat in the first exon of the FMR-1 gene, which induces hypermethylation of the CpG island in the promoter region results in the transcriptional suppression of FMR-1 gene. Besides, several deletions and point mutation in the FMR-1 gene resulting in fragile X syndrome have been reported. Conventionally, cytogenetic method was used to diagnose fragile X syndrome. However, this method is time- consuming and not accurate. The current diagnostic method, Southernblot analysis, gives a more reliable result, but, it is also very time-consuming. To develop a more rapid, economic, and accurate assay system, we tried RT-PCR. From 32 fragile X syndrome suspects, the absence of FMR-1 expression was found in 3 of them.Southern blot analysis confirmed that that inactivation of FMR-1 gene expressionis indeed due to the mutation of FMR-1 gene. Thus far, the application of RT-PCRon prenatal diagnosis demonstrated that RT-PCR is useful to detect FMR-1 mRNA in normal male fetus. Our studies indicated that this assay can be used to diagnose the fragile X syndrome for the majority of male patients. By constant endeavor, wehope that RT-PCR can be routinely used in clinical diagnosis.