本文關(guān)鍵詞： 遺傳性疾病 無(wú)纖維蛋白原血癥 基因突變 剪接位點(diǎn)　出處：《華北理工大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

【摘要】：目的對(duì)一個(gè)遺傳性無(wú)纖維蛋白原血癥的先證者及其家系進(jìn)行基因分析,篩選致病基因突變,并探討該家系發(fā)病的可能分子機(jī)制。方法1常規(guī)實(shí)驗(yàn)室方法檢測(cè)先證者及其胞弟、父母凝血功能中活化部分凝血活酶時(shí)間(APTT)、凝血酶原時(shí)間(PT)、凝血酶時(shí)間(TT),通過(guò)免疫比濁法及凝固法(cluss法)分別檢測(cè)纖維蛋白原抗原及其活性。2免疫印跡實(shí)驗(yàn)(Western-blot)檢測(cè)家系成員及正常對(duì)照者外周血血漿纖維蛋白原α鏈、β鏈及γ鏈3條肽鏈的表達(dá)。3提取家系成員及健康對(duì)照者外周血基因組DNA,普通PCR擴(kuò)增編碼纖維蛋白原3條肽鏈的3個(gè)基因FGA、FGB、FGG的所有外顯子、外顯子與內(nèi)含子交界處、啟動(dòng)子區(qū)序列,通過(guò)一代測(cè)序?qū)ふ铱赡艿闹虏』蛲蛔儭Ｍㄟ^(guò)二代測(cè)序技術(shù)對(duì)先證者及其母親進(jìn)行全基因組測(cè)序,篩選與引起疾病有關(guān)的基因突變。篩選的基因突變與NCBI基因數(shù)據(jù)庫(kù)比較排除基因多態(tài)性。4將發(fā)現(xiàn)的可能致病基因突變拷貝入神經(jīng)元網(wǎng)絡(luò)剪接位點(diǎn)預(yù)測(cè)軟件預(yù)測(cè)基因突變可能引起的剪接位點(diǎn)改變。分別以先證者及對(duì)照者外周血DNA為模板,設(shè)計(jì)引物擴(kuò)增包含突變位置的突變序列及正常序列,分別克隆至哺乳動(dòng)物載體pc DNA3.1(-),構(gòu)建含有突變序列的FGG小基因質(zhì)粒(Wt-FGG)及含有正常序列的FGG小基因質(zhì)粒(NtFGG),并將2種質(zhì)粒分別轉(zhuǎn)染COS-7細(xì)胞,提取細(xì)胞總RNA,逆轉(zhuǎn)錄PCR(RT-PCR)法逆轉(zhuǎn)錄成c DNA。經(jīng)瓊脂糖凝膠電泳初步驗(yàn)證目的片段,并進(jìn)行一代測(cè)序,將測(cè)序結(jié)果與NCBI中正常m RNA序列比對(duì)。利用DNASIS軟件分析基因突變后可能引起的氨基酸改變。結(jié)果1先證者及胞弟APTT200s、PT及TT100s,父母的檢測(cè)值均正常。免疫比濁法及Cluss法檢測(cè)先證者及胞弟纖維蛋白原含量均為0,父母的檢測(cè)值均略低于正常值下限。2 Western-blot法檢測(cè)先證者及胞弟外周血無(wú)纖維蛋白原肽鏈表達(dá),父母與正常對(duì)照者分別在65KD、52KD、46KD顯示3條帶,為α鏈、β鏈及γ鏈,但父母纖維蛋白原肽鏈表達(dá)低于對(duì)照者。3一代測(cè)序未在外顯子與內(nèi)含子交界處及啟動(dòng)子區(qū)發(fā)現(xiàn)基因突變,外顯子區(qū)僅在FGA的5號(hào)外顯子第4266位核苷酸檢測(cè)到基因突變,先證者及胞弟核苷酸g4266 A純合突變?yōu)镚,使第331位蘇氨酸錯(cuò)義突變?yōu)楸彼?P 331 ThrAla),父母為相同位置的雜合突變,該突變是已經(jīng)報(bào)道的基因多態(tài)性位點(diǎn)。對(duì)先證者及其母親二代全基因組測(cè)序后,經(jīng)過(guò)篩選,先證者FGG 3號(hào)內(nèi)含子第5個(gè)核苷酸G純合突變?yōu)锳,FGG IVS3+5GA,母親為相同突變位置的雜合子,通過(guò)一代測(cè)序驗(yàn)證其他家系成員相同位置,胞弟與先證者為相同的純合突變,父親與母親為相同位置的雜合突變,檢測(cè)30名正常健康對(duì)照者未發(fā)現(xiàn)相同的基因突變,查詢NCBI中SNP基因數(shù)據(jù)庫(kù)排除基因位點(diǎn)多態(tài)性。4剪接位點(diǎn)預(yù)測(cè)軟件結(jié)果示突變后序列供體剪接位點(diǎn)消失,未激活隱蔽剪接位點(diǎn)及產(chǎn)生新的剪接位點(diǎn)。以先證者及健康對(duì)照者外周血DNA為模板擴(kuò)增的片段長(zhǎng)766bp,Wt-FGG逆轉(zhuǎn)錄后的c DNA經(jīng)瓊脂糖凝膠電泳目的條帶約280bp,Nt-FGG的c DNA約為460bp,一代測(cè)序驗(yàn)證結(jié)果顯示W(wǎng)t-FGG m RNA與Nt-FGG m RNA相比缺少3號(hào)外顯子,共184個(gè)堿基。與NCBI中FGG m RNA氨基酸序列比對(duì),突變后形成的異常m RNA缺少3號(hào)外顯子所編碼的第16至76個(gè)氨基酸。DNASIS軟件顯示在4號(hào)外顯子的第2個(gè)氨基酸形成終止密碼子,預(yù)測(cè)體外構(gòu)建的細(xì)胞模型轉(zhuǎn)錄后形成只含16個(gè)氨基酸的截短γ鏈。結(jié)論1 FGG基因3號(hào)內(nèi)含子的第5個(gè)核苷酸G突變?yōu)锳是該家系遺傳性無(wú)纖維蛋白原血癥的致病基因,該突變?yōu)閲?guó)內(nèi)首次報(bào)道的致病基因突變。2剪接位點(diǎn)突變后導(dǎo)致異常m RNA剪接,3號(hào)外顯子跳讀形成截短的γ鏈,是該家系纖維蛋白原不能正常合成的可能發(fā)病機(jī)制。
[Abstract]:Objective to afibrinogenemia of a hereditary proband and family gene analysis, gene mutation screening, and to explore the possible molecular mechanisms of the pathogenesis of this family. The 1 detection methods of routine laboratory methods of the proband and his younger brother, parents in the blood coagulation function of activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), by immune turbidimetry and solidification method (cluss method) were used to detect the fibrinogen antigen and activity of.2 Western blot (Western-blot) detection of plasma fibrinogen in peripheral blood of family members and controls the original alpha chain, the expression of.3 beta chain and gamma chain 3 peptides extraction of family members and controls the peripheral blood genomic DNA, DNA encoding fibrinogen of 3 peptides of 3 genes FGA, FGB PCR, FGG all exons, exon intron junction, the sequence of the promoter region,. A generation of candidate genes for sequencing mutations. Through the two generation sequencing technology on the proband and his mother of whole genome sequencing, screening and disease related gene mutation screening. The gene mutation between NCBI gene and.4 gene polymorphism database exclusion will find may be pathogenic gene mutation is copied into a neural network of splice sites the prediction software predicted gene splice site mutations may cause the change. In the proband and control of peripheral blood DNA as template, amplified sequence contains mutations and normal sequence positions of primers were designed respectively to clone mammalian vector PC DNA3.1 (-), FGG gene plasmid construct containing small mutation sequence (Wt-FGG) and with normal sequence FGG gene plasmid (NtFGG), and 2 kinds of plasmids were transfected into COS-7 cells, extracted RNA, reverse transcription PCR (RT-PCR) was transcribed into C DNA. by June Fat sugar gel electrophoresis preliminary verification of target fragment, and generation sequencing, the sequencing results in NCBI and normal m RNA sequence using DNASIS software. The analysis of amino acid mutations may cause the change. Results of the 1 probands and brother of APTT200s, PT and TT100s, their detection values were normal. Immune turbidity detection method and Cluss method, the proband and brother of fibrinogen content was 0, the detection value of parents were slightly lower than normal value detection limit.2 Western-blot proband and brother of peripheral blood without fibrinogen peptide expression, parents and normal controls respectively in 65KD, 52KD, 46KD showed 3 bands, for alpha beta and gamma chain chain chain, but the parents of fibrinogen peptide expression was lower than that of control.3 generation sequencing in the exon intron junction and the promoter region found mutations in exon 4266th only exon nucleotide detection in exon 5 to FGA Mutations in the proband and his younger brother g4266 nucleotide A homozygous mutation of G, the 331st missense mutation to alanine threonine (P 331 ThrAla), the parents for the same position of heterozygous mutation, the mutation gene polymorphism has been reported. The proband and his mother of two generation of whole genome sequencing. After screening, the proband FGG 3 intron fifth nucleotide G homozygous mutation of A, FGG IVS3+5GA, mother of the same heterozygous mutations in the same position, verify the position of other family members through generation sequencing, and brother of the proband was the same homozygous mutation, the father and the mother for the same position heterozygous mutation detection, 30 healthy controls were not found in the same gene mutation, SNP gene database query NCBI exclude polymorphism.4 splice site prediction software showed mutation sequence the donor splice site did not disappear, activation of cryptic splice Site and produce new splicing sites. The proband and healthy control peripheral blood DNA were amplified fragment length 766bp, Wt-FGG reverse transcription C after DNA by agarose gel electrophoresis band of about 280bp, Nt-FGG C DNA is about 460bp, the next-generation sequencing results show that Wt-FGG m RNA and Nt-FGG m RNA lacks exon 3, a total of 184 bases in M RNA and NCBI FGG. The amino acid sequence alignment, abnormal m RNA mutation formed after missing exon 3 encoding sixteenth amino acids to 76.DNASIS termination codon software that formed in the 4 exon of second amino acids, the transcription of cell model in vitro after formation of truncated gamma chain containing only 16 amino acids. The fifth nucleotide G conclusion 1 FGG gene intron 3 to A mutation is the causative gene of the family of congenital afibrinogenemia, the mutation was first reported in China's disease Mutation in the.2 gene splice site mutation leads to aberrant m splicing RNA, gamma chain exon 3 skipping form truncated, this family is not normal fibrinogen synthesis mechanisms.

【學(xué)位授予單位】：華北理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R596.1

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