本文關(guān)鍵詞： 先天性白內(nèi)障 CRYBB2基因 基因突變 聚合酶鏈反應(yīng) 限制性片段長(zhǎng)度多態(tài)性　出處：《浙江大學(xué)》2011年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：目的： 先天性白內(nèi)障是兒童期視力缺損的首要病因之一。遺傳突變是最常見(jiàn)的致病因素。迄今為止,至少已發(fā)現(xiàn)與遺傳性的先天性白內(nèi)障相關(guān)的24個(gè)疾病相關(guān)基因。本實(shí)驗(yàn)對(duì)一個(gè)后極性表型的先天性白內(nèi)障家系進(jìn)行疾病相關(guān)候選基因定位。 方法： 本實(shí)驗(yàn)擬研究一四代的先天性白內(nèi)障大家系,共27人,患者9名。所有家系成員均接受病史調(diào)查和眼科檢查,包括視力、裂隙燈、散瞳眼底檢查等。抽取外周血樣本,提取基因組DNA。聚合酶鏈反應(yīng)(PCR)擴(kuò)增相關(guān)的候選基因的編碼區(qū)及內(nèi)含子、外顯子交界區(qū)。擴(kuò)增產(chǎn)物雙向測(cè)序,并進(jìn)行序列分析。限制性片段長(zhǎng)度多態(tài)性(RFLP)進(jìn)一步驗(yàn)證突變。SIFT, PolyPhen, Align-GVGD三種計(jì)算法和疏水性分析預(yù)測(cè)突變對(duì)蛋白質(zhì)結(jié)構(gòu)、功能的影響。 結(jié)果： 裂隙燈下檢查該家系表型為后極性。分析家系圖,遺傳方式為常染色體顯性遺傳。測(cè)序結(jié)果發(fā)現(xiàn)CRYBB2基因編碼區(qū)一條等位基因的第5位堿基有C—T改變,導(dǎo)致了第2位氨基酸由高保守的丙氨酸變?yōu)槔i氨酸(CRYBB2-A2V).該堿基改變于9名患者中共分離,而不存在于27名健康家系成員及100名健康者對(duì)照。RFLP證實(shí)該突變致使患者丟失一個(gè)HaeⅢ酶切位點(diǎn),而健康的家庭成員和100個(gè)健康者對(duì)照中未有改變。SIFT, PolyPhen, Align-GVGD分?jǐn)?shù)均提示A2V為可影響蛋白質(zhì)結(jié)構(gòu)和功能的有意義的突變。疏水性分析結(jié)果顯示A2V突變蛋白在突變區(qū)域的疏水性高于野生型β-晶狀體蛋白。 結(jié)論： 本研究首次證實(shí)了CRYBB2基因第2外顯子中第5位堿基發(fā)生C—T改變是與后極性先天性白內(nèi)障表型共分離的錯(cuò)義突變,CRYBB2基因?yàn)楹髽O性先天性白內(nèi)障的疾病相關(guān)候選基因。進(jìn)一步證實(shí)了先天性白內(nèi)障的遺傳異質(zhì)性,以及白內(nèi)障表型與基因型的相對(duì)相關(guān)性,支持了βB2-晶狀體蛋白在人晶狀體發(fā)育及白內(nèi)障形成過(guò)程中具有重要作用的理論。
[Abstract]:Objective:. Congenital cataract is one of the leading causes of childhood visual impairment. Genetic mutation is the most common cause. At least 24 disease-related genes associated with inherited congenital cataract have been identified. Methods:. In this study, we studied 14 and 4 generations of congenital cataract, including 27 patients and 9 patients. All the family members underwent medical history investigation and ophthalmologic examination, including visual acuity, slit lamp, mydriasis fundus examination, etc. Peripheral blood samples were taken. Genomic DNA.Polymerase chain reaction (PCR) was used to amplify the coding region, intron and exon junctions of the candidate genes. Restriction fragment length polymorphism (RFLP) further verified the effect of mutation SIFT, PolyPhen, Align-GVGD and hydrophobic analysis on protein structure and function. Results:. The phenotype of the pedigree was examined to be post polarity under slit lamp. The genetic pattern was autosomal dominant inheritance. The results of sequencing showed that the fifth base of an allele in the coding region of CRYBB2 gene had C-T changes. The result was that the second amino acid changed from a highly conserved alanine to a valine, CRYBB2-A2V, which was isolated from nine patients. RFLP did not exist in 27 healthy family members and 100 healthy controls. RFLP confirmed that the mutation resulted in the loss of a Hae 鈪，

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