本文選題：粘脂累積病 + 先天性眼球震顫　； 參考：《華中科技大學(xué)》2012年博士論文

【摘要】：本文收集并鑒定了一個粘脂累積病及兩個先天性眼球震顫家系,通過連鎖分析(Linkage Analysis)與定位克隆(Positional Cloning)分子遺傳學(xué)技術(shù)對這三個家系進(jìn)行了遺傳學(xué)分析,確定了它們的致病原因。同時對一個功能不清楚的眼球震顫致病基因FRMD7進(jìn)行了初步的功能分析。其結(jié)果如下： 1、粘脂累積病家系的分子遺傳學(xué)分析 對收集和鑒定的一對夫婦(非近親婚配)生育了三個粘脂累積病孩子(兩女一男)進(jìn)行微衛(wèi)星標(biāo)記的連鎖分析,發(fā)現(xiàn)GNPTG基因附近的微衛(wèi)星標(biāo)記D16S3024和選擇對先證者GNPTG基因所有外顯子和外顯子／內(nèi)含子交界處進(jìn)行直接測序,發(fā)現(xiàn)了先征者GNPTG基因存在兩個未報導(dǎo)的雜合突變(即一個復(fù)合型雜合突變)IVS4-1GC(剪切突變)和c.471de1C(單堿基缺失突變)。對其雙親測序發(fā)現(xiàn),先征者攜帶的一個雜合IVS4-1GC突變是來自其母親,而另一個雜合的c.471de1C缺失突變是來自父親。同時對另外的兩位患者進(jìn)行上述兩個突變位置的測序檢測,發(fā)現(xiàn)這兩位患者攜帶與先征者相同的突變。；通過體外和體內(nèi)反轉(zhuǎn)錄-聚合酶鏈?zhǔn)椒磻?yīng)(RT-PCR)及測序?qū)嶒?發(fā)現(xiàn)IVS4-1GC突變導(dǎo)致增加第4內(nèi)含子74個堿基的InRNA的產(chǎn)生,該mRNA翻譯的GNPTG蛋白在第4個外顯子編碼開始處產(chǎn)生一個終止密碼子,因此也產(chǎn)生一個可能無功能的蛋白。說明GNPTG基因的IVS4-1GC和c.471de1C這個復(fù)合型雜合突變是導(dǎo)致該家系三個患者的發(fā)病原因。 2、X連鎖先天性眼球震顫家系的分子遺傳學(xué)分析 收集并鑒定了兩個X-連鎖的先天性眼球震顫家系。家系1的遺傳方式為X-連鎖隱性遺傳,通過微衛(wèi)星標(biāo)記的連鎖分析,發(fā)現(xiàn)不能排除GPR143基因突變是導(dǎo)致該家系眼球震顫的原因,對先證者GPR143基因所有外顯子和外顯子／內(nèi)含子交界處直接測序,在該基因編碼框819到822位的四個堿基(AACA)缺失(c.819822de1),這是一個新的突變,該突變造成GPR143蛋白在274位移碼,移碼7個氨基酸后產(chǎn)生一個終止密碼子(p.T274Ifs*8),產(chǎn)生一個截短蛋白；運(yùn)用單鏈構(gòu)象多態(tài)性(SSCP)分析,家系中所有男性患者和女性攜帶者都存在該突變,而家系外100例(50例男性和50女性)正常人不存在該突變。家系2的遺傳方式為X-連鎖顯性遺傳,先證者及其母親眼球震顫并伴隨頭震顫和斜視等癥狀,根據(jù)這些臨床癥狀不能排除FRMD7基因突變所致；因此選擇先征者基因組DNA,對FRMD7基因所有外顯子和外顯子交界處直接測序,在該基因的580位發(fā)現(xiàn)單個堿基改變即c.580GA(p.A194T)；并且該突變存在男性患者和女性患者,且女性患者為雜合突變,不存在于100例(50例男性和50女性)正常人群；通過生物信息學(xué)氨基酸保守性分析,發(fā)現(xiàn)194位的丙氨酸從線蟲到人類都是高度保守的。以上研究推測GPR143基因p.T274IfsX7突變和FRMD7基因c.580GA(p.A194T)突變分別是導(dǎo)致眼球震顫家系1和家系2的致病原因。3、眼球震顫致病基因FRMD7初步的功能分析 為了初步探討FRMD7如何參與細(xì)胞分化功能,本研究構(gòu)建了FRMD7野生型、突變體p.A194T、p.H333fs(本實(shí)驗室之前所發(fā)現(xiàn)突變)和p.G24R(文獻(xiàn)中報道的突變)分別與EGFP形成融合蛋白載體,觀察各類突變體和野生型在真核細(xì)胞中定位情況,發(fā)現(xiàn)突變體p.A194T和野生型類似,主要定位于細(xì)胞質(zhì),而突變體p.G24R和p.H333fs發(fā)生核轉(zhuǎn)位現(xiàn)象,絕大部分融合蛋白集中在細(xì)胞核中。通過生物信息學(xué)預(yù)測NLS位于FRMD7氨基酸序列的234位KRKH,NES位于93位的LTRYLFTLQI。并進(jìn)行了功能驗證。為了進(jìn)一步分析FRMD7蛋白入核后的功能,本實(shí)驗分析了其與p53蛋白的共定位分析,結(jié)果沒有發(fā)現(xiàn)明顯的共定位情況；另外通過免疫熒光技術(shù)證明FRMD7在核內(nèi)過表達(dá)形成的點(diǎn)狀結(jié)構(gòu)也不是細(xì)胞核常見結(jié)構(gòu)Cajal body。說明FRMD7對于神經(jīng)元細(xì)胞分化的促進(jìn)作用可能不是通過與p53蛋白及Cajal body實(shí)現(xiàn)的。 總之,本研究主要是以粘脂累積病及先天性眼球震顫家系為對象,通過分子遺傳學(xué)及分子生物學(xué)等手段,分析一個粘脂累積病家系和兩個先天性眼球震顫的致病原因,同時對一個眼球震顫基因FRMD7蛋白亞細(xì)胞定位的可能機(jī)制進(jìn)行了初步探討。在粘脂累積病家系發(fā)現(xiàn)的致病基因GNPTG的雜合突變是首次在中國人群中檢測到的基因變異,擴(kuò)充了該基因的突變譜；這一研究結(jié)果可以將該疾病與其癥狀相近(粘多糖累積�、�、粘脂累積�、驛)的其他疾病準(zhǔn)確區(qū)分開來,有利于實(shí)現(xiàn)對該家系病人的個體化治療方案的制定。對于X連鎖的先天性眼球震顫家系1的研究結(jié)果再一次支持了在中國人群中,GPR143基因的突變所導(dǎo)致的先天性眼球震顫疾病與在白種人群中表現(xiàn)的I型白化病不同。對于X連鎖的先天性眼球震顫家系2的研究結(jié)果擴(kuò)展了眼球震顫致病基因FRMD7的突變譜。這些遺傳學(xué)研究結(jié)果對于疾病的準(zhǔn)確分型至關(guān)重要,是實(shí)施個體化針對性治療的基礎(chǔ),而且可以作為遺傳病家系產(chǎn)前診斷的遺傳學(xué)基礎(chǔ)。對于FRMD7蛋白功能的初步分析結(jié)果則提示了部分FRMD7的突變所導(dǎo)致的亞細(xì)胞定位的改變可能是導(dǎo)致蛋白在細(xì)胞質(zhì)中的功能喪失的主要原因。這一結(jié)果有助于加深對FRMD7蛋白突變的致病機(jī)制的認(rèn)識。
[Abstract]:In this paper , we collected and identified a myxosis and two congeners of ocular tremor , and analyzed these three families by linkage analysis and molecular genetic technique . The pathogenic factors were determined . At the same time , a preliminary functional analysis was carried out on a functional unknown pathogenic gene FRMD7 . The results were as follows :
1 . Molecular genetic analysis of myxolipid accumulation disease
Two unrelated heterozygous mutations ( i.e . , a compound heterozygous mutation ) IVS4 - 1GC ( shear mutation ) and c . 471de1C ( single base deletion mutation ) were detected by the microsatellite markers D16S3024 in the vicinity of GNPTG gene .
In vitro and in vivo reverse transcription - polymerase chain reaction ( RT - PCR ) and sequencing experiments , it was found that the IVS4 - 1GC mutation resulted in an increase in the production of InRNA of 74 bases in the 4th intron , which translated GNPTG proteins at the beginning of the 4th exon coding to produce a stop codon , thus also producing a protein that may not function . This compound heterozygous mutation of the GNPTG gene IVS4 - 1GC and c.471de1C is the cause of the three patients in the family .
Molecular genetic analysis of 2 , X - linked congenital exophthalmos
The genetic pattern of family 1 was X - linked recessive inheritance , and the linkage analysis of microsatellite markers found that it was not possible to exclude the gene mutation of the gene . The deletion of the four bases ( AACA ) in the gene coding block 819 to 822 was a new mutation which resulted in a stop codon ( p.T274Ifs * 8 ) at the position 274 of the gene encoding the amino acids of the gene coding block 819 to 822 , resulting in a truncated protein .
Single - strand conformation polymorphism ( SSCP ) analysis showed that the mutation was found in all male and female carriers in the family , but there were 100 cases ( 50 males and 50 females ) in the family without the mutation .
Therefore , the genomic DNA of the precursor was selected and sequenced directly at the junction of all exon and exon of the FRMD7 gene , and a single base change , i.e . , c.580 GA ( p . A194T ) was found at 580 bits of the gene ;
and the mutation existed in male and female patients , and female patients were heterozygous mutations , and did not exist in 100 cases ( 50 males and 50 females ) in the normal population ;
Through the analysis of amino acid conservation in bioinformatics , it was found that the 194 - position alanine was highly conserved from nematodes to humans . The above studies have speculated that the mutation of the p . T274Ifs7 mutation and the c . 580 GA ( p . A194T ) mutation of the gene of the pH.580 GA ( p . A194T ) , respectively , are responsible for the pathogenesis of the ocular tremor family 1 and the family 2 . 3 . The preliminary functional analysis of the pathogenic gene FRMD7 of the eyeball tremor
In order to investigate the role of FRMD7 in cell differentiation , we constructed FRMD7 wild - type , mutant p . A194T , p . H333fs ( mutation identified earlier in the laboratory ) and p . G24R ( the mutation reported in the literature ) . We observed the localization of various mutants and wild type in eukaryotic cells .
In addition , the dot - like structure formed by overexpression of FRMD7 in the nucleus by immunofluorescence technique is not a common structure of the nucleus of the nucleus . It is suggested that the promotion of FRMD7 to neuronal cell differentiation may not be achieved by interaction with p53 protein and CaSO body .
In conclusion , this study was mainly focused on the pathogenesis of myxolipid accumulation disease and congenital eyeball tremor , and the possible mechanism for the localization of a myxomil gene FRMD7 protein was investigated by molecular genetics and molecular biology . The mutation of GNPTG gene was the first to be detected in Chinese population , which extended the mutation spectrum of the gene .
The results of this study can distinguish the disease from other diseases closely related to its symptoms ( mucopolysaccharidosis 鈪，

本文編號：2013028