人類印痕區域11p15.5遺傳疾病之分析

Analysis of Human Imprinting gene 11p15.5

楊錦雯(Chin-Wen Yang)

指導教授 ： 李銘仁

國立臺灣大學/醫學院/分子醫學研究所/碩士(2008年)

https://doi.org/10.6342/NTU.2008.10319

摘要

與人類印痕區域11p15.5相關的甲基化異常遺傳疾病包含貝克威思-威德曼症候群 （Beckwith-Wiedemann syndrome）及羅素-西弗症候群（Russell-Silver syndrome）。導致此兩種疾病發生的主要原因，是由於染色體印痕區域11p15.5上的LIT1與H19基因甲基化程度發生異常。本論文的研究主題有二：(1)開發簡單快速之檢測系統用以偵測甲基化程度的變化（主要是利用甲基化敏感性限制酶結合定量聚合酶連鎖反應），並(2)將此一新開發技術，用於貝克威思-威德曼症候群及羅素-西弗症候群患者LIT1與H19基因座上的DNA甲基化程度檢測。在檢測10名貝克威思-威德曼症候群病患、20名羅素-西弗症候群的病患及20名健康個體LIT1基因的甲基化程度結果中，在健康個體所獲得的基因甲基化指數（Methylation Index; MI）平均值±2個標準差為53.9 %± 14.61（正常參考值：39.29 %~68.51 %）；而在10名貝克威思-威德曼症候群患者的基因甲基化指數中，有五位屬於基因低度甲基化，甲基化指數分別為8.42 %、8.47 %、7.35 %、19.04 %及4.48 %，平均值±2個標準差為9.59 ± 11.08，四位的甲基化指數落於正常參考值範圍內，其值分別為55.28 %、51.34 %、55.87 %及57.8 %；而有ㄧ位甲基化程度介於正常值與低度甲基化之間，甲基化指數為36.7 %，推測這可能與遺傳鑲嵌（genetic mosaicism）現象有關。在羅素-西弗症候群患者裡，LIT1甲基化指數均落在正常參考值範圍內，顯示這些病患可能與LIT1基因甲基化異常無關。在檢測H19基因甲基化時，發現在健康個體所測得的甲基化指數不符合預期(其值均呈現明顯低度甲基化，MI<<1)，以軟體分析經聚合酶連鎖反應擴增後的單股DNA序列發現，分子會形成明顯的二級結構，推測這可能會影響引子黏合（annealing）效率，進而致使所偵測的甲基化指數與預期結果不符；因此，學生目前初步所開發的E-Q-PCR暫時還無法應用於H19基因的甲基化程度檢測。然而，本研究所開發的新技術已證實在LIT1基因座上可以快速準確偵測其甲基化程度變化，此外，本方法只須少量DNA即可進行甲基化程度檢測，因此可將其發展應用於產前篩檢上。

關鍵字

人類印痕區域11p15.5 ； 甲基化 ； 甲基化敏感性限制酶結合即時定量聚合酶連鎖反應 ； 貝克威思-威德曼症候群 ； 羅素-西弗症候群

並列摘要

The imprinting regions at chromosome11p15.5 are associated with Beckwith-Wiedemann syndrome (BWS) and Russell-Silver syndrome (RSS). The most frequent findings are the defect of methylation on the imprinting loci LIT1 and H19, both located at chromosome11p15.5, in patients with BWS and RSS. Therefore this study is to develop a simplified and high-performance method for assessment of the degrees of DNA methylation. The novel method is designed by coupling the methylation-sensitive endonuclease treatment and the quantitative polymerase chain reaction (called E-Q-PCR). And the method was employed to evaluate the variable of methylation on the imprinting loci LIT1 and H19. A total of 10 BWS patients, 20 RSS patients and 20 healthy individuals were analysed. The methylation index (MI) of the 20 health individuals was estimated to be ranged from 39.29 %~68.51 % (mean ± 2 SD = 53.9% ± 14.61) which was arbitrarily designated as the normal range of the MI. For the ten BWS patients, five reveal LIT1 hypomethylation with the MI values, 8.42 %, 8.47 %, 7.35 %, 19.04 % and 4.48 % (mean ± 2 SD = 9.59 % ± 11.08 ), four show normal LIT1 methylation pattern, MI= 55.28 %, 51.34 %, 55.89 % and 57.8 %, (mean ± 2 SD = 55.08 % ± 5.42), and one has the MI value (MI= 36.7 %) between the normal and hypomethylation ranges, possibly caused by a genetic mosaicism. For the 20 RSS patients, neither hypomethylation nor hypermethylation was identified. I noted unexpected results were obtained when E-Q-PCR was performed on the H19 locus which might be due to the secondary structure of the single strand DNA produced during PCR. Despite this, E-Q-PCR remains to be a practical method for estimating degrees of DNA methylation since it has been successfully applied on the LIT1 locus. This molecular approach deserves more effort to further characterize its sensitivity, specificity, and cost-effective analyses when compared with other molecular diagnostic techniques.

並列關鍵字

imprinting ； methylation ； methylation-sensitive endonuclease-coupled quantitative polymerase chain reaction (E-Q-PCR) ； Beckwith-Wiedemann syndrome ； Russell-Silver syndrome

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