先天性甲狀腺功能低下症病人甲狀腺過氧化脢之分子分析

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題名:	先天性甲狀腺功能低下症病人甲狀腺過氧化脢之分子分析 Molecular Analysis of Thyroid Peroxidase (TPO) in Patients with Congenital Hypothyroidism
作者:	鄔哲源
貢獻者:	中國醫藥學院醫學研究部
關鍵詞:	先天性甲狀腺機能不足;甲狀腺過氧化脢？;選殖;移碼突變;錯義突變;碘有機化缺失;Congenital hypothyroidism;Thyroid peroxidase (TPO);Cloning;frameshift mutation;Missense mutation;Total iodide organification defect
日期:	2001-07
上傳時間:	2010-09-06 23:34:52 (UTC+8)
摘要:	甲狀腺過氧化脢(基因名稱為TPO)為甲狀腺荷爾蒙合成之主要酵素，TPO 基因的缺失為造成因碘有機化缺失造成之先天性甲狀腺功能低下症之主要原因，本計劃寄望能藉由TPO 基因的分析，釐清先天性甲狀腺功能低下症之分子基礎。其他種族(荷蘭人，巴西人，日本人及Amish 人)的甲狀腺功能低下症之遺傳研究顯示，大部份的TPO 突變均落於TPO 基因的exon 2, 8, 9, 10 及14 上，過去一年藉由直接定序該五個exons，我們在總共有二十個TPO alleles 中確認了兩個不同的新突變。該兩突變均為一個核甘酸之缺損。此研究結果顯示台灣先天性甲狀腺功能低下症病人之TPO 突變分佈與其他族群並不相同，我們寄望更深入的研究能找出大部份的TPO 基因突變，建立起國人的核酸資料庫。我們所提計劃，將包括下列項目：1. 將人類之全長TPO 互補去氧核醣核酸克隆(Clone)進一個可在哺乳細胞中表現之載體。2. TPO 基因的突變分析：(1) 南方點漬分析：用以找出大片斷缺失突變 (2) 北方點漬分析：用以找出RNA 轉錄突變 (3) 單股多型性分析及直接核酸定序分析我們利用單股多型性分析先篩檢所有TPO 基因之exon，然後再利用直接核酸定序分析，針對有異常單股多型性之片段，找出核甘酸之改變。3. 功能研究針對所有因核酸改變而導致之錯譯突變，我們將其核酸改變導入正常之TPO 互補去氧核醣核酸(cDNA)中，該TPO cDNA 置於哺乳細胞表現載體，並過渡轉殖入COS-7細胞，測量甲狀腺過氧化脢之活性，藉以觀測突變效應。開發出分子診斷方法，以便快速診斷出TPO 基因之突變；如此將有助於產前分子診斷暨隱性帶原者之確認。 Thyroid peroxidase (TPO) is the key enzyme in the synthesis of thyroid hormone. Defects in the TPO gene were reported to be the main cause of congenital hypothyroidism due to a total iodide organification defect. This defect is the most common hereditary inborn errors causing congenital hypothyroidism. In this grant proposal, we plan to elucidate the molecular basis of hereditary congenital hypothyroidism caused by total iodination defect through TPO gene. Mutation studies in Dutch, Brazilian, Japanese, and Amishes indicated that most of the TPO mutation fell into exons 2, 8, 9, 10, and 14 of TPO gene. In the past year, we have identified two different mutant alleles in TPO gene out of 20 alleles analyzed by direct sequencing the above five exons. Both mutations were not reported in other ethnic populations and were novel. They both were one nucleotide deletion, resulting in frameshift mutation. The preliminary data indicated that the distribution of TPO mutation is heterogeneous among different ethnic populations. We think our preliminary findings justify for more in-depth study of TPO gene. Our proposed study for the next year include the following: I. Cloning of human full-length cDNA of TPO into a mammalian expression vector . II. To elucidate the molecular defects of TPO gene in congenital hypothyroidism patients. (1) Southern blot analysis to identify large DNA fragment deletion. (2) Nor thern blot analysis to identify mutation that will affect mRNA transcript either in quantity or in size. (3) Single strand conformation polymorphism (SSCP) analysis and direct DNA sequencing. After Southern and Northern blot analyses, SSCP will be used first to screen all the 17 exons of TPO and direct sequencing will be performed for those showing abnormal shift in SSCP analysis. III. Functional analysis of those identified novel nucleotide changes in TPO gene. All the nucleotide changes that will lead to missense mutation will be put into a normal TPO cDNA, which was constructed in a mammalian expression vector, such as pcDNA3. The resulting plasmid will be transfected into COS-7 cells by transient transfection and activity of thyroid peroxidase will be assayed to evaluate the mutation effect. IV. Development of rapid molecular diagnosis method for TPO mutation detection. After most TPO mutations are identified, amplification created restriction site method will be developed for rapid molecular diagnosis of TPO mutation. This will allow for future prenatal diagnosis and car r ier assessment.
顯示於類別:	[醫學研究所] 研究計畫

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