本文選題：先天性白內(nèi)障　切入點(diǎn)：基因突變　出處：《浙江大學(xué)》2014年博士論文

【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。遺傳突變是最常見的致病因素。迄今為止,已發(fā)現(xiàn)與遺傳性先天性白內(nèi)障相關(guān)約37條致病基因、數(shù)百個突變位點(diǎn)。本實(shí)驗(yàn)對兩個繞核性表型的先天性白內(nèi)障家系進(jìn)行疾病相關(guān)候選基因定位及功能研究。 方法： 本實(shí)驗(yàn)擬研究在浙江大學(xué)附屬第二醫(yī)院眼科中心收集的兩個先天性白內(nèi)障家系。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)物雙向測序,并進(jìn)行序列分析。SIFT, PolyPhen-2和疏水性分析預(yù)測突變對蛋白質(zhì)結(jié)構(gòu)、功能的影響。 從人眼cDNA文庫中獲得目的基因,通過DNA重組技術(shù)克隆至真核表達(dá)載體pEGFP-N1,構(gòu)建野生型質(zhì)粒。利用定點(diǎn)突變技術(shù)構(gòu)建攜帶突變基因的質(zhì)粒。將野生組(Cx46wt-EGFP)、突變組質(zhì)粒(Cx46H95Y-EGFP、Cx46R76H-EGFP)及空白質(zhì)粒(pEGFP-N1)轉(zhuǎn)染入Hek293細(xì)胞,G418抗生素篩選,建立穩(wěn)定轉(zhuǎn)染細(xì)胞系,觀察蛋白亞細(xì)胞定位和縫隙連接形成情況。在低鈣、生理鈣及縫隙連接通道抑制劑環(huán)境下,運(yùn)用極性染料染核技術(shù),檢測野生組與突變組半通道功能。 結(jié)果： 裂隙燈下檢查兩個家系表型皆為繞核性。分析家系圖,遺傳方式均為常染色體顯性遺傳。測序結(jié)果：家系一：發(fā)現(xiàn)CX46基因編碼區(qū)一條等位基因的第283位堿基由胞嘧啶變?yōu)樾叵汆奏?c.283CT),導(dǎo)致了第95位氨基酸由高保守的組氨酸變?yōu)槔野彼?p.H95Y)。家系二：發(fā)現(xiàn)CX46基因編碼區(qū)一條等位基因的第227位堿基由鳥嘌呤變?yōu)橄汆堰?c.227GA),導(dǎo)致了第76位氨基酸由高保守的精氨酸變?yōu)榻M氨酸(p.R76H)。這兩個堿基改變在相應(yīng)的家系患者中共分離,而不存在于健康的家族成員及對照組100名中國漢族健康者。SIFT, PolyPhen-2均提示Cx46-R76H、H95Y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H95Y突變蛋白在突變區(qū)域的疏水性高于野生型縫隙連接蛋白46,而R76H突變蛋白疏水性低于野生型。 在突變的功能研究部分,我們成功建立了穩(wěn)定轉(zhuǎn)染Cx46wt-EGFP, Cx46H95Y-EGFP, Cx46R76H-EGFP, pEGFP-N1質(zhì)粒的Hek293細(xì)胞系。在野生組和H95Y突變組細(xì)胞中都能觀察到縫隙連接的形成,但H95Y突變組的縫隙連接數(shù)量明顯少于野生組。而R76H突變組極少觀察到縫隙連接的形成。Cx46H95Y-EGFP, Cx46R76H-EGFP兩組突變細(xì)胞質(zhì)中均可見高熒光聚集。在半通道功能檢測中,當(dāng)?shù)外}環(huán)境下,野生組細(xì)胞因有正�？p隙連接半通道的存在,可攝取PI和DAPI兩種染料,而Cx46H95Y-EGFP, Cx46R76H-EGFP突變組和pEGFP-N1空白組細(xì)胞不能攝取；當(dāng)在生理鈣和縫隙連接蛋白通道抑制劑環(huán)境下,四組細(xì)胞均不能攝取染料。 結(jié)論： 本研究發(fā)現(xiàn)了與繞核性先天性白內(nèi)障相關(guān)的兩個CX46基因錯義突變：其中,c.283CT為首次報道的新突變,c.227GA為已報道的突變。Cx46蛋白H95Y和R76H突變可導(dǎo)致蛋白胞質(zhì)內(nèi)聚集,降低縫隙連接的表達(dá),影響縫隙連接半通道的正常開放。本研究拓展了先天性白內(nèi)障的基因突變譜,揭示了縫隙連接蛋白突變導(dǎo)致先天性白內(nèi)障發(fā)病的機(jī)制,為先天性白內(nèi)障的預(yù)防和基因治療提供理論基礎(chǔ)。
[Abstract]:Purpose :

Congenital cataract is one of the primary causes of visual impairment in children . Genetic mutations are the most common pathogenic factors . So far , about 37 pathogenic genes and hundreds of mutation sites associated with hereditary congenital cataract have been found .

Method :

In this study , two congenital cataract families collected at the Second Affiliated Hospital of Zhejiang University were studied . All of the members received medical history investigation and ophthalmologic examination , including vision , slit lamp , mydriatic fundus examination and so on . Peripheral blood samples were taken to extract genomic DNA . Polymerase chain reaction ( PCR ) was used to amplify the coding region of candidate genes and intron and exon boundary region . The amplification products were sequenced in two directions and sequenced . The effects of mutation on protein structure and function were analyzed by SIFT , PolyPhen - 2 and hydrophobic analysis .

The target gene was obtained from human eye cDNA library , and the wild type plasmid was constructed by cloning the plasmid containing the mutant gene by DNA recombination technique . The wild group ( Cx46wt - EGFP ) , the mutant group plasmid ( Cx46H95Y - EGFP , Cx46R76H - EGFP ) and the blank plasmid were transfected into Hek293 cells .

Results :

The results of the sequencing showed that the 283rd base of one allele of CX46 gene coding region was changed from cytosine to adenine ( c.283CT ) .

In this study , we successfully established Hek293 cell line stably transfected with Cx46wt - EGFP , Cx46H95Y - EGFP , Cx46R76H - EGFP and eukaryotic expression plasmids .
When in physiological calcium and gap junction protein channel inhibitor environment , four groups of cells do not uptake dye .

Conclusion :

The mutation of Cx46 protein H95Y and R76H can lead to intracellular accumulation of protein , decrease the expression of gap junction and affect the normal opening of gap junction half - channel . This study extends the gene mutation spectrum of congenital cataract , and reveals the mechanism of connexin mutation leading to congenital cataract . It provides a theoretical basis for preventing and gene therapy of congenital cataract .

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R776.1

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 鄭建秋,馬志偉,孫慧敏;中國東北漢族一個先天性白內(nèi)障家系致病基因的鑒定[J];中華醫(yī)學(xué)遺傳學(xué)雜志;2005年01期

，

本文編號：1672143