發(fā)布時(shí)間：2018-06-04 22:29

  本文選題：SCN1A基因突變 + 剪接　； 參考：《廣州醫(yī)科大學(xué)》2014年碩士論文

【摘要】：【目的】 選取熱性驚厥相關(guān)性癲癇患者SCN1A基因突變，通過生物信息學(xué)軟件分析其對剪切的影響，構(gòu)建mini-gene，進(jìn)行體外剪接分析；并探討特異性U1snRNA及Morpholino修復(fù)異常剪接的機(jī)理。 【對象和方法】 選取本實(shí)驗(yàn)室篩查出的熱性驚厥相關(guān)性癲癇患者SCN1A基因突變(c.473+5GA,c.909AG, c.3705+1GT)為研究對象，用Human Splicing Finder2.4軟件進(jìn)行預(yù)測c.473+5GA和c.909AG突變體的異常剪接情況（其中c.3705+1GT的異常剪接情況在本課題組既往研究已明確）。構(gòu)建mini-gene（pTARGET-Exon2-3-4-5，pTARGET-Exon5-6-7-8）轉(zhuǎn)染HEK293細(xì)胞，提取總RNA，進(jìn)行RT-PCR,分析異常剪接的情況。 根據(jù)上述三個(gè)突變位點(diǎn)設(shè)計(jì)與剪接供體位點(diǎn)完全互補(bǔ)或與其附近內(nèi)含子區(qū)，外顯子區(qū)多個(gè)靶位點(diǎn)互補(bǔ)的特異性U1snRNA表達(dá)載體；與野生型或突變型mini-gene（1:1）共轉(zhuǎn)染HEK293細(xì)胞，，提取總RNA，進(jìn)行RT-PCR，分析正常與異常轉(zhuǎn)錄本比例，比較特異性U1snRNA修復(fù)異常剪接的效果。同時(shí)對突變c.909AG設(shè)計(jì)與其新激活的剪接供體位點(diǎn)互補(bǔ)的Morpholino寡核苷酸，分析對異常剪接的修復(fù)效果，并與其U1snRNA靶向修復(fù)的效果對比。每組實(shí)驗(yàn)重復(fù)三次，獲得均值，計(jì)量資料是由均數(shù)±標(biāo)準(zhǔn)差（X±S)表示，使用SPSS16.0軟件包進(jìn)行統(tǒng)計(jì)分析。兩樣本均數(shù)比較采用t檢驗(yàn)。以P 0.05具有統(tǒng)計(jì)學(xué)意義。 【結(jié)果】 1. SCN1A基因突變體外剪切分析結(jié)果 PEFS+患者c.473+5GA突變體異常轉(zhuǎn)錄本較正常的缺少98bp堿基，即SCN1A基因的2號外顯子5'端8bp堿基和全3號外顯子的缺失。FS+患者c.909AG突變體異常轉(zhuǎn)錄本較正常的缺少60bp堿基，即6號外顯子5'端60bp堿基缺失。兩種異常剪切結(jié)果與軟件預(yù)測結(jié)果一致。 2.特異性U1snRNA修復(fù)異常剪切的效果 （1） c.473+5GA的mini-gene突變體，表達(dá)正常轉(zhuǎn)錄本占總轉(zhuǎn)錄本（異常轉(zhuǎn)錄本和正常轉(zhuǎn)錄本的和）的比例為35.37%±3.29%。與特異性U1snRNA(E3Mu,E3+1, E3+9, E3+16)共轉(zhuǎn)染后，正常轉(zhuǎn)錄本比例增加至59.88%±4.46%，54.94%±1.97%，100%，100%。它們與單純突變體轉(zhuǎn)染時(shí)正常轉(zhuǎn)錄本比例相比，均有統(tǒng)計(jì)學(xué)差異(p0.05)。 （2）c.909AG的mini-gene突變體，表達(dá)正常轉(zhuǎn)錄本占總轉(zhuǎn)錄本比例為30.34%±2.71%。與特異性U1snRNA(E6+1, E6+9, E6+16, E6-9, E6-18)共轉(zhuǎn)染后，正常轉(zhuǎn)錄本比例分別為92.85%±2.08%,100%,100%,27.47%±1.80%,29.83%±3.54%。與單純突變體的正常轉(zhuǎn)錄本比例相比，E6+1, E6+9, E6+16有統(tǒng)計(jì)學(xué)差異(p0.05)；E6-9, E6-18無統(tǒng)計(jì)學(xué)差異(p0.05)。 （3）c.3705+1GT的突變體mini-gene（產(chǎn)生兩個(gè)異常轉(zhuǎn)錄本，即SCN1A基因的18號外顯子5'端缺失49bp堿基或者18號內(nèi)含子5'端保留20bp堿基），表達(dá)大片段異常轉(zhuǎn)錄本占總轉(zhuǎn)錄本的比例為37.53%±2.27%。與特異性U1snRNA(E18Mu, E18+1, E18+9, E18+16)共轉(zhuǎn)染后，表達(dá)大片段異常轉(zhuǎn)錄本比例分別為55.49%±1.38%，64.66%±3.64%，46.39%±2.14％，80.29%±3.51%，與單純突變體的相比，有統(tǒng)計(jì)學(xué)差異(p0.05)，但無正常轉(zhuǎn)錄本的表達(dá)。 3. Morpholino寡核苷酸的修復(fù)異常剪切的效果 c.909AG的mini-gene突變體，表達(dá)正常轉(zhuǎn)錄本占總轉(zhuǎn)錄本比例為30.75%±2.25%，與Morpholino（終濃度分別為10uM，50uM）共轉(zhuǎn)染后，正常轉(zhuǎn)錄本比例分別為41.64%±1.94%，64.54%±2.47%，27.24%±2.72%；與單純突變體的相比，有統(tǒng)計(jì)學(xué)差異(p0.05)。 【結(jié)論】 1.SCN1A基因c.437+5GA和c.909AG突變，可能通過影響剪切與疾病的發(fā)生相關(guān)。 2.特異性U1snRNA對不同位置的突變修復(fù)效果不一樣，其中剪接供體位點(diǎn)高度保守區(qū)+1位置的突變修復(fù)效果最差；結(jié)合于不同靶位點(diǎn)的特異性U1snRNA修復(fù)效果不同，結(jié)合于內(nèi)含子區(qū)的修復(fù)效果較好。 3.特異性U1snRNA和Morpholino對異常剪切都有修復(fù)作用，但U1snRNA的修復(fù)效果較好。
[Abstract]:[Objective]
The SCN1A gene mutation in epileptic patients with febrile seizures was selected, and the effects on shear were analyzed by bioinformatics software. The mini-gene was constructed and the splicing was analyzed in vitro, and the mechanism of abnormal splicing of specific U1snRNA and Morpholino was discussed.
[object and method]
The SCN1A gene mutation (c.473+5GA, c.909AG, c.3705+1GT) in patients with febrile convulsion related seizures (c.909AG, c.3705+1GT) was selected as the research object. The abnormal splicing of c.473+5GA and c.909AG mutants was predicted with Human Splicing Finder2.4 software (in which the abnormal splicing of c.3705+1GT was clearly defined in our previous research group). Mini-gene (pTARGET-Exon2-3-4-5, pTARGET-Exon5-6-7-8) was transfected into HEK293 cells, total RNA was extracted, RT-PCR was analyzed, and abnormal splicing was analyzed.
According to the above three mutation sites, the specific U1snRNA expression vector was complementation with the splicing site or its nearby introns, and the target loci of the exons were complementing. The HEK293 cells were co transfected with the wild type or mutant mini-gene (1:1), and the total RNA was extracted, and the ratio of normal to abnormal transcriptional transcript was analyzed, and the ratio of normal to abnormal transcriptional transcript was comparatively specific. The effect of abnormal splicing was repaired by sexual U1snRNA. At the same time, the Morpholino oligonucleotides complementing the mutation c.909AG design with the newly activated splice donor sites were analyzed and compared with the effect of the U1snRNA targeted repair. Each group of experiments repeated three times and obtained the mean value. The measurement data were mean standard deviation (X + S) table. SPSS16.0 software package was used for statistical analysis. Two samples were compared with t test. P 0.05 was statistically significant.
[results]
The results of in vitro shear analysis of 1. SCN1A gene mutations
The abnormal transcriptional transcript of the c.473+5GA mutant in PEFS+ patients is less than the normal 98bp base, that is, the 8bp base of the SCN1A gene 2 exon 5'terminal 8bp base and the total 3 exon deletion.FS+ patient c.909AG mutant abnormal transcriptional transcript is less than normal 60BP base, that is, the 60BP base deficiency at the 5' end of exon 6. Two abnormal shear results and software prediction results. Agreement.
The effect of 2. specific U1snRNA in the repair of abnormal shear
(1) the mini-gene mutant of c.473+5GA, which expressed the proportion of normal transcriptional transcripts in the total transcriptional transcript and normal transcript, was 35.37% + 3.29%. and specific U1snRNA (E3Mu, E3+1, E3+9, E3+16) Co transfected, and the normal transcriptional ratio increased to 59.88% + 4.46%, 54.94% + 1.97%, 100%, and 100%., and they were transfected with the simple mutant. The proportion of frequent transcripts was statistically different (P0.05).
(2) the mini-gene mutant of c.909AG, the normal transcriptional transcript was co transfected with the total transcriptional ratio of 30.34% + 2.71%. and specific U1snRNA (E6+1, E6+9, E6+16, E6-9, E6-18), the normal transcriptional ratio was 92.85% + 2.08%, 100%, 100%, 27.47% + 1.80%, 29.83% + 3.54%. compared to the normal transcriptional ratio of the simple mutant, E6+1, E6+9. 6+16 had statistical difference (P0.05); E6-9 and E6-18 had no statistical difference (P0.05).
(3) the mutant mini-gene of c.3705+1GT (producing two abnormal transcripts, namely the 5'terminal deletion 49bp base of the exon 18 of the SCN1A gene or the 20bp base in the 5' end of the intron 18), expressed the proportion of the large fragment of the abnormal transcriptional book in the total transcript of 37.53% + 2.27%. and the specific U1snRNA (E18Mu, E18+1, E18+9, and E18+9). The proportion of abnormal transcripts was 55.49% + 1.38%, 64.66% + 3.64%, 46.39% + 2.14% and 80.29% + 3.51% respectively. Compared with the simple mutant, there were statistical differences (P0.05), but there was no normal transcriptional transcript.
Effect of 3. Morpholino oligonucleotide repair on abnormal shearing
The normal transcriptional version of the mini-gene mutant of c.909AG was 30.75% + 2.25%, and the normal transcriptional ratio was 41.64% + 1.94%, 64.54% + 2.47% and 27.24% + 2.72%, respectively, with Morpholino (final concentration 10uM, 50uM). Compared with the simple mutant, there was a statistically significant difference (P0.05).
[Conclusion]
The mutation of c.437+5GA and c.909AG of 1.SCN1A gene may be related to the occurrence of disease through shear.
The effect of 2. specific U1snRNA on mutation repair at different locations is different, in which the mutation repair effect of the splice donor site at the highly conserved +1 location is the worst, and the effect of the specific U1snRNA repair combined with the different target loci is different, and the repair effect is better in the intron in the intron.
3. specific U1snRNA and Morpholino can repair abnormal shear, but U1snRNA has better repair effect.
【學(xué)位授予單位】：廣州醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R742.1

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 章國衛(wèi),宋懷東,陳竺;mRNA選擇性剪接的分子機(jī)制[J];遺傳學(xué)報(bào);2004年01期

本文編號：1979098