一个遗传性无纤维蛋白原血症家系的基因缺陷分析

发布时间：2018-01-26 07:25

本文关键词： 遗传性疾病无纤维蛋白原血症基因突变剪接位点　出处：《华北理工大学》2016年硕士论文　论文类型：学位论文

【摘要】：目的对一个遗传性无纤维蛋白原血症的先证者及其家系进行基因分析,筛选致病基因突变,并探讨该家系发病的可能分子机制。方法1常规实验室方法检测先证者及其胞弟、父母凝血功能中活化部分凝血活酶时间(APTT)、凝血酶原时间(PT)、凝血酶时间(TT),通过免疫比浊法及凝固法(cluss法)分别检测纤维蛋白原抗原及其活性。2免疫印迹实验(Western-blot)检测家系成员及正常对照者外周血血浆纤维蛋白原α链、β链及γ链3条肽链的表达。3提取家系成员及健康对照者外周血基因组DNA,普通PCR扩增编码纤维蛋白原3条肽链的3个基因FGA、FGB、FGG的所有外显子、外显子与内含子交界处、启动子区序列,通过一代测序寻找可能的致病基因突变。通过二代测序技术对先证者及其母亲进行全基因组测序,筛选与引起疾病有关的基因突变。筛选的基因突变与NCBI基因数据库比较排除基因多态性。4将发现的可能致病基因突变拷贝入神经元网络剪接位点预测软件预测基因突变可能引起的剪接位点改变。分别以先证者及对照者外周血DNA为模板,设计引物扩增包含突变位置的突变序列及正常序列,分别克隆至哺乳动物载体pc DNA3.1(-),构建含有突变序列的FGG小基因质粒(Wt-FGG)及含有正常序列的FGG小基因质粒(NtFGG),并将2种质粒分别转染COS-7细胞,提取细胞总RNA,逆转录PCR(RT-PCR)法逆转录成c DNA。经琼脂糖凝胶电泳初步验证目的片段,并进行一代测序,将测序结果与NCBI中正常m RNA序列比对。利用DNASIS软件分析基因突变后可能引起的氨基酸改变。结果1先证者及胞弟APTT200s、PT及TT100s,父母的检测值均正常。免疫比浊法及Cluss法检测先证者及胞弟纤维蛋白原含量均为0,父母的检测值均略低于正常值下限。2 Western-blot法检测先证者及胞弟外周血无纤维蛋白原肽链表达,父母与正常对照者分别在65KD、52KD、46KD显示3条带,为α链、β链及γ链,但父母纤维蛋白原肽链表达低于对照者。3一代测序未在外显子与内含子交界处及启动子区发现基因突变,外显子区仅在FGA的5号外显子第4266位核苷酸检测到基因突变,先证者及胞弟核苷酸g4266 A纯合突变为G,使第331位苏氨酸错义突变为丙氨酸(P 331 ThrAla),父母为相同位置的杂合突变,该突变是已经报道的基因多态性位点。对先证者及其母亲二代全基因组测序后,经过筛选,先证者FGG 3号内含子第5个核苷酸G纯合突变为A,FGG IVS3+5GA,母亲为相同突变位置的杂合子,通过一代测序验证其他家系成员相同位置,胞弟与先证者为相同的纯合突变,父亲与母亲为相同位置的杂合突变,检测30名正常健康对照者未发现相同的基因突变,查询NCBI中SNP基因数据库排除基因位点多态性。4剪接位点预测软件结果示突变后序列供体剪接位点消失,未激活隐蔽剪接位点及产生新的剪接位点。以先证者及健康对照者外周血DNA为模板扩增的片段长766bp,Wt-FGG逆转录后的c DNA经琼脂糖凝胶电泳目的条带约280bp,Nt-FGG的c DNA约为460bp,一代测序验证结果显示Wt-FGG m RNA与Nt-FGG m RNA相比缺少3号外显子,共184个碱基。与NCBI中FGG m RNA氨基酸序列比对,突变后形成的异常m RNA缺少3号外显子所编码的第16至76个氨基酸。DNASIS软件显示在4号外显子的第2个氨基酸形成终止密码子,预测体外构建的细胞模型转录后形成只含16个氨基酸的截短γ链。结论1 FGG基因3号内含子的第5个核苷酸G突变为A是该家系遗传性无纤维蛋白原血症的致病基因,该突变为国内首次报道的致病基因突变。2剪接位点突变后导致异常m RNA剪接,3号外显子跳读形成截短的γ链,是该家系纤维蛋白原不能正常合成的可能发病机制。
[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.

【学位授予单位】：华北理工大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R596.1

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