儿童免疫性血小板减少症与FV10、VWF28、GP1BA基因变异相关性实验探索与分析

发布时间：2018-04-05 00:24

  本文选题：免疫性血小板减少症　切入点：凝血因子V　出处：《广西医科大学》2012年硕士论文

【摘要】：目的探讨儿童免疫性血小板减少症(ITP)发病及病情与血小板膜糖蛋白(GP1BA)基因、FV因子第10外显子(FV10)、VWF基因第28外显子(VWF28)变异相关性,以求对明确儿童ITP发病与此基因变异有无的遗传相关性,探索ITP发病的遗传机制。 方法对51例临床诊断为儿童ITP的患儿和28例非ITP患儿的外周血进行全基因组DNA提取,参照CCDS数据库人类基因序列设计PCR引物,以基因组DNA为模板对GP1BA、FV10、VWF28基因进行PCR扩增、凝胶电泳、基因测序,依据NCBI等数据库信息整理两组数据进行统计学分析。 结果(1)GP1BA基因存在T161M,(24.0%,5.97%,P=0.011)、W614R氨基酸变异点,检测样本基因存在大段缺失现象,发现GP1BA基因有重复串联现象。(2)成功扩增实验组及对照组所有患儿FV10基因；与数据库FV基因及氨基酸序列比结果显示ITP组和对照组均存在氨基酸R513K变异(94.1%、100%,P=0.191),杂合子率为87.5%、78.6%(P=0.303)；两组氨基酸均存在Q534R变异,且变异率为100%；未发现FV氨基酸506位的FVLeiden突变(核苷酸G1691A变异\氨基酸A506G变异)；(3)成功扩增90%以上样本VWF28基因全序列,发现V1229I、V1229G杂合、N1231T杂合、G1253S杂合、W1313R杂合、T1381A变异(约78%)、D1472H杂合、E1376G杂合、V1565L氨基酸变异共9种氨基酸变异。GCC---TTG脱氧核糖核酸具有连锁遗传特性,V1565L的变异不论是纯合状态还是杂合状态,100%都会与A1555A(GCC、GAA) (?)勺杂合碱基无义突变连锁出现； 结论(1)GP1AB基因上的T161M的变异与儿童ITP的发生相关。(2)GP1BA的W614R变异,可能与患儿ITP的发生有关。(3)FV发生的R513K、Q534R氨基酸变异与儿童ITP发生无相关性。(4)ITP发生与FV Lieden突变无相关性。(5)儿童ITP的发生可能与VWF28基因变异有关；VWF28上V1229I,V1229G,N1231T,G1253S,W1313R,E1376G等变异,可能与患儿ITP的发生有关。T1381A、D1472H变异、E1376G变异与儿童ITP发生无关。(6)广西地区人口VWF28基因存在GCC---TTG脱氧核糖核酸的连锁遗传；(7)部分儿童ITP发生与基因变异有关；尚不能确定儿童ITP与FV、VWF、GPIBA全基因变异的相互作用。
[Abstract]:Objective to investigate the relationship between the pathogenesis and condition of ITPs in children with immune thrombocytopenia and the variation of VWF28 in exon 10 of FV10 exon 10 of platelet membrane glycoprotein (GP1BA) gene.In order to find out the genetic correlation between ITP and the gene variation in children, and to explore the genetic mechanism of ITP.Methods Genomic DNA was extracted from peripheral blood of 51 children with clinically diagnosed ITP and 28 children without ITP. PCR primers were designed according to the human gene sequence of CCDS database. Genomic DNA was used as template for PCR amplification of GP1 BAFV10VWF28 gene.Gel electrophoresis, gene sequencing, NCBI and other database information collate the two groups of data for statistical analysis.Results the amino acid mutation of W614R was detected at 5.97 and 5.97. The FV10 gene of all the children in the experimental group and the control group was amplified successfully. The results showed that there was a large deletion in the sample gene and the GP1BA gene was duplicated and connected with the other two genes in the experiment group and in the control group, and the FV10 gene of all the children in the experimental group and the control group was amplified successfully.Compared with FV gene and amino acid sequence in database, the amino acid R513K mutation was found in ITP group and control group. The amino acid R513K mutation was 94.1and 0.191%, the heterozygote rate was 87.5% and 78.6% (P < 0.05), Q534R mutation was found in both groups.There was no FVLeiden mutation (nucleotide G1691A mutation / amino acid A506G mutation) at position 506 of FV amino acid to amplify the complete sequence of VWF28 gene in more than 90% of the samples.鍙戠幇V1229I,V1229G鏉傚悎,N1231T鏉傚悎,G1253S鏉傚悎,W1313R鏉傚悎,T1381A鍙樺紓(绾，

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