利用變性高效能液相層析偵測肢帶型肌失養症2B亞型病人之DYSF基因突變分析

Mutation analysis of the DYSF gene in LGMD2B patients using DHPLC

鄭獻文(Hsien-Wen Cheng)

指導教授 ： 鐘育志

高雄醫學大學/醫學院/醫學研究所/碩士(2007年)

摘要

轉譯dysferlin蛋白質之DYSF gene常見發生突變於三好氏肌營養不良症(Miyoshi myopathy，MM）以及肢帶型肌失養症2B亞型（Limb girdle muscular dystrophy 2B，LGMD 2B），此兩種病皆為dysferlin蛋白質缺失導致之症候群。dysferlin蛋白質缺失症候群主要診斷特徵為肌肉纖維肌膜（sarcolemma）上dysferlin蛋白質缺失並在DYSF gene上發生突變。DYSF gene，位在染色體2q13上，並且其包含55 exons更含括150 kb genomic DNA。我們分別在11位無親戚相關之LGMD 2B病人進行基因分析，其中7位為疑似LGMD2B台灣病人，4位日本LGMD2B病人已知帶有6個突變點位在DYSF gene上。所有病人在肌肉上都呈現dysferlin蛋白質缺失或大幅減少。利用變性高效能液相層析（DHPLC）針對基因之55 exons的PCR products做初步篩選，並在找尋出序列突變後利用核酸定序加以確認。在7個台灣病人，總共在DYSF gene上確認出17個序列改變，其中3個會造成單一胺基酸改變並且為新突變，另外 14個則為已知的單核酸多形性(Single nucleotide polymorphism)。這三個新發現之突變，G486，A3472G，C334T，因為該突變在150正常人偵測並未發現相似圖形即可推測與致病相關(SNP<1%)。這三個突變為散佈在整個基因序列。

關鍵字

肢帶型肌失養症2B亞型(LGMD 2B) ； Dysferlin基因 ； 變性高效能液相層析(DHPLC)

並列摘要

Dysferlin（DYSF） gene encoding dysferlin is mutated in Miyoshi myopathy and Limb-Girdle Muscular Dystrophy type 2B（LGMD 2B）, the two main phenotypes recognized in dysferlinopathies. Dysferlin deficiency in muscle is the most relevant feature for the diagnosis of dysferlinopathy and prompts the search for mutations in DYSF gene. DYSF, located on chromosome 2p13, contains 55 coding exons and spans 150 kb of genomic DNA. We performed a genomic analysis of the DYSF coding sequence in 11 unrelated LGMD 2B patients, including 7 suspected LGMD 2B Taiwanese patients, and 4 Japanese patients with 6 confirmed mutations in DYSF gene. All patients showed an absence or drastic decrease of dysferlin expression in muscle. A primary screening of DYSF using denaturing high performance liquid chromatography（DHPLC）of PCR products of each of 55 exons of the gene was followed by sequencing whenever a variation was detected. In 7 Taiwanese patients, 17 sequence variations were identified in DYSF, 3 of which predict single amino-acid substitution and are novel, while the other 14 changes are known SNPs (Single nucleotide polymorphisms). The three novel sequence variations, G486、A3472G、C334T, was not detected in 150 normal individuals and was identified as potential pathogenic mutation (SNP<1%). These three mutations were widely spread in the coding sequence of the gene.

並列關鍵字

Limb-Girdle Muscular Dystrophy type 2B(LGMD 2B ； Dysferlin(DYSF) gene、Denaturing high performance liquid chromatography(DHPLC)

參考文獻

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