基于基因表达谱的儿童噬血性淋巴组织细胞增生症候选基因筛选及相关实验研究

发布时间：2018-03-04 08:18

  本文选题：噬血性淋巴组织细胞增生症　切入点：儿童　出处：《广西医科大学》2015年博士论文　论文类型：学位论文

【摘要】：第一部分基于基因表达谱的儿童噬血性淋巴组织细胞增生症候选基因筛选及相关实验研究目的应用生物信息学方法,筛选新的儿童噬血性淋巴组织细胞增生症(He-mophagocytic lymphohistiocytosis, HLH)候选疾病基因并进行实验验证,探讨儿童HLH发病和预后的分子机制。方法(1)从基因表达综合数据库(Gene expression omnibus, GEO)中下载GSE26050儿童HLH基因芯片数据集,然后在R语言软件包中进行差异表达基因(Differentially expressed genes, DEGs)分析,将获得的DEGs定义为“检测基因集”；采用Genecards和Fable文献挖掘已知儿童HLH疾病基因,将其定义为“训练基因集”；最后,利用Toppgene在线分析工具筛选出儿童HLH候选疾病基因。(2)以100例HLH患儿为病例组,146例健康儿童为对照组,采集外周静脉血,采用多重聚合酶链式反应(Polymerase chain reaction, PCR)和SNaPshot技术对部分候选基因单核苷酸多态性(Single nucleotide polymorphism, SNP)进行检测。(3)用Haploview v.4.2软件进行各SNP位点哈-温平衡(Hardy-Weinberg eq-uilibrium, HWE)和相互间的连锁不平衡(Linkage disequilibrium, LD)检验。用SPSS17.0软件分析各位点基因型频率、等位基因频率分布差异与儿童HLH发病的相关性。风险度采用比值比(Odds ratio, OR)及其95%可信区间(Confidence interval,CI)表示。用PHASE2.1软件进行单体型的构建和分析。P小于0.05视为差异有统计学意义。(4)对入选HLH患儿进行病历资料收集并随访。以确诊HLH为观察起点,死亡日期作为观察终点,失访、末次随访时仍存活(包括缓解、疾病活动、疾病复发的患儿)的病例则确定为截尾病例。以有意义的SNPs不同基因型作为研究因素,采用Kaplan-Meier曲线进行生存分析,应用Log-rank检验比较生存曲线。以P0.05为差异有统计学意义。结果(1)获得一个含86个基因的“检测基因集”和一个含73个基因的“训练基因集”；采用Toppgene共获得45个儿童HLH候选疾病基因,其中表达上调的基因42个,表达下调的基因3个,为TNFRSF17、CX3CR1和CCR2。(2)TNFRSF17基因多态性分析结果：病例组rs3743591GG基因型频率高于对照组,差异有统计学意义(10.0% vs.1.4%,P=0.002),提示rs3743591GG基因型是儿童HLH发病的易感因素(OR=8.000,95% CI=1.714-37.349);病例组rs3743591G等位基因频率高于对照组,差异有统计学意义(20.5% vs.10.6%,P=0.002),提示rs3743591G等位基因是儿童HLH发病的易感因素(OR=2.171,95% CI=1.308-3.603)。病例组rs2017662AA基因型频率高于对照组,差异有统计学意义(11.0% vs.1.4%,P=0.001),提示rs2017662AA基因型是儿童HLH发病的易感因素(OR=8.899,95% CI=1.928-41.082);病例组rs2017662A等位基因频率高于对照组,差异有统计学意义(21.0% vs.10.3%,P=0.001),提示rs2017662A等位基因是儿童HLH发病的易感因素(OR=2.322,95% 0=1.396-3.860)。(3) CX3CR1基因多态性分析结果：病例组rs2853712TT基因型频率高于对照组,差异有统计学意义(72.0% vs.50.0%,P=0.001),提示rs2853712TT基因型是儿童HLH发病的易感因素(OR=2.571,95% CI=1.493-4.430)。(4)CCR2基因多态性分析结果：病例组rs1799865TT基因型频率高于对照组,差异有统计学意义(39.0% vs.20.5%,P=0.002),提示rs1799865TT基因型是儿童HLH发病的易感因素(OR=2.472,95% CI=1.401-4.363);病例组rS 1799865T等位基因频率高于对照组,差异有统计学意义(53.0% vs.37.0%,P=0.000),提示rs1799865T等位基因是儿童HLH发病的易感因素(OR=1.921,95% CI=1.333-2.769)。(5)单体型分析结果：单体型A-T-G-G-G-C-T-T-T-T-T-A-G-A-C (SNPs顺序：rs3743591-rs11570151-rs2017662-rs2071336-rs2669850-rs17793056-rs13088991-rs13062158-rs2853712-rs2669841-rs2853711-rs3762823-rs309296 3-rs3092962-rsl 799865)在病例组出现的频率高于对照组,差异有统计学意义(5.7% vs.1.3%,P=0.003),提示单体型A-T-G-G-G-C-T-T-T-T-T-A-G-A-C是儿童HLH发病的易感因素(OR=5.025,95% CI=1.530-16.505).单体型G-T-A-G-G-T-C-C-T-C-G-A-G-A-C (SNPsJl顺序：rs3743591-rs11570151-rs2017662-rs2071336-rs2669850-rs17793056-rs13088991-rs13062158-rs28537 12-rs2669841-rs2853711-rs3762823-rs3092963-rs3092962-rs 1799865)在病例组出现的频率高于对照组,差异有统计学意义(5.2%VS.0%,P=4.43×10-5),提示单体型G-T-A-G-G-T-C-C-T-C-G-A-G-A-C是儿童HLH发病的易感因素。(6)儿童HLH病例中,携带rs3743591GG基因型者中位生存时间小于携带rs3743591AA/AG基因型者,差异有统计学意义(86.0个月vs.118.0个月,P=0.009);携带rs2017662AA基因型者中位生存时间小于携带rs2017662GG/GA基因型者,差异有统计学意义(88.0个月vs.118.0个月,P=0.004)；携带rs2853712TT基因型者平均生存时间与携带rs2853712CC/CT基因型者比较差别无统计学意义(96.5个月vS.108.6个月,P=0.124)；携带rs1799865TT基因型者中位生存时间小于携带rs1799865CC/CT基因型者,差异有统计学意义(92.0个月vs.107.0个月,P=0.021)。结论(1)本研究应用生物信息学方法筛选出45个新的儿童HLH候选疾病基因,包括42个上调表达基因和3个下调表达基因。(2) TNFRSF17、CX3CR1和CCR2基因在儿童HLH中表达下调,提示其可能参与儿童HLH的发病机制。(3)TNFRSF17基因rs3743591、rs2017662, CX3CR1基因rs2853712, CCR2基因rs1799865多态位点与儿童HLH发病有关。(4) TNFRSF17基因、CX3CR1基因和CCR2基因多态位点构成的单体型A-T-G-G-G-C-T-T-T-T-T-A-G-A-C和G-T-A-G-G-T-C-C-T-C-G-A-G-A-C与儿童HLH发病有关。(5) TNFRSF17基因rs3743591、rs2017662, CCR2基因rs1799865多态位点有可能作为儿童HLH预后判断的分子标记。A-T-G-G-G-C-T-T-T-T-T-A-G-A-C和G-T-A-G-G-T-C-C-T-C-G-A-G-A-C与儿童HLH发病有关。第二部分儿童噬血性淋巴组织细胞增生症SH2D1A、XIAP基因变异和临床意义目的了解SH2区蛋白1A (SH2 domain protein 1A, SH2D1A)基因和X连锁凋亡抑制蛋白(X-linked inhibitor of apoptosis protein, XIAP)基因突变和序列变异在HLH患儿中的发生情况,探讨SH2D1A、XIAP基因变异与儿童HLH的关系。方法病例组为100例HLH患儿,对照组为100例健康儿童。采集外周静脉血,提取基因组DNA,采用PCR法扩增SH2D1A基因1-4外显子和XIAP基因1-6外显子编码区及其侧翼内含子区片段,PCR产物经ABI PRISM3730全自动DNA测序仪直接测序,然后与NCBI数据库中SH2D1A和XIAP基因的序列进行比对,获得基因变异信息,利用生物信息学方法预测SH2D1A和XIAP基因突变对蛋白质功能的影响。对基因突变的病例回顾性分析其临床资料,将其临床表型与基因型进行对比分析。结果(1)在HLH病例组中发现XIAP基因1个新的错义突变,30544 TA,位于第1外显子上,导致所编码的氨基酸发生改变,Phe27Ile。该位点突变在对照组中未发现,突变发生率为1%(1/100)。(2)用PolyPhen2和SIFT软件预测XIAP基因错义突变功能,显示XIAP基因Phe27Ile突变有可能影响蛋白质功能。(3)在病例组和对照组中共发现5个SNPs位点,包括SH2D1A基因的rs72610640、rs182286559多态和XIAP基因的rs5956583、rs140997240、rs28382740多态；在病例组和对照组中比较了这5个SNPs位点的基因型、等位基因的频率分布,差异均无统计学意义,提示这些SNPs位点可能与儿童HLH发病无关。(4)临床特征分析：发生Phe27Ile突变的1例患儿无明显HLH阳性家族史和特殊既往史,13岁起病,患儿临床表现为HLH,按照HLH-2004方案接受化疗曾经得到缓解,但停药后复发,治疗效果差,预后不良,于16岁时死于多器官功能衰竭(Multiple system organ failure, MSOF)。结论本研究发现了1个新的XIAP基因错义突变,Phe27Ile,该突变可能与HLH患儿的发生和不良预后有关。
[Abstract]:The first part is based on the gene expression of macrophage lymphoid tissue cell syndrome children's bloody hyperplasia spectrum selection of gene screening and related experiments using bioinformatics methods, screening of children with bloody bite lymphoid tissue cell hyperplasia (He-mophagocytic lymphohistiocytosis, new HLH) experiments were carried out to test the candidate disease gene and explore the molecular mechanisms of the pathogenesis and prognosis of children with HLH. Methods (1) the expression of gene from the integrated database (Gene Expression Omnibus, GEO) in the download GSE26050 HLH gene chip data sets, and then the gene differential expression in the R language software package (Differentially expressed, genes, DEGs) analysis, the definition of DEGs is obtained for detection of gene set mining HLH gene known diseases "; children with Genecards and Fable documents, which is defined as" training gene set "; finally, analysis tools were screened by Toppgene online. Tong HLH gene candidate disease. (2) in 100 HLH patients as case group, 146 healthy children as control group, peripheral venous blood, using multiplex polymerase chain reaction (Polymerase chain reaction, PCR) and SNaPshot technology on the part of the candidate gene single nucleotide polymorphism (Single nucleotide polymorphism, SNP) were detected. (3) using Haploview v.4.2 software for the SNP locus of Hardy Weinberg equilibrium (Hardy-Weinberg eq-uilibrium, HWE) and the inter linkage disequilibrium (Linkage disequilibrium LD) test. Each genotype frequency was analyzed with the SPSS17.0 software, the correlation of allele frequency distribution and incidence of pediatric HLH risk with odds ratio. (Odds ratio, OR) and 95% confidence interval (Confidence, interval, CI). Construction and analysis of.P is less than 0.05 as there were statistically significant differences in haplotype with PHASE2.1 software (4) of the selected. HLH children with medical records collected and diagnosed by HLH were followed up. The date of death as starting point, end point, lost, alive at the final follow-up (including remission, disease activity, disease recurrence) cases were identified as censored cases. The meaning of different genotypes of SNPs as the study factor, the the Kaplan-Meier curve of survival analysis, using Log-rank test to compare the survival curves with P0.05. The difference was statistically significant. Results (1) obtained a 86 genes "gene sets and a 73 gene" training gene set "; using Toppgene received a total of 45 children with HLH disease candidate gene. The expression of 42 genes, 3 downregulated genes, TNFRSF17, CX3CR1 and CCR2. (2) TNFRSF17 gene polymorphism analysis results: the case group rs3743591GG genotype frequency was higher than the control group, the difference was statistically The significance of (10% vs.1.4%, P=0.002), suggesting that rs3743591GG genotype is susceptible children factor for the onset of HLH (OR=8.000,95% CI=1.714-37.349); rs3743591G allele frequency in case group was higher than the control group, the difference was statistically significant (20.5% vs.10.6%, P=0.002), suggesting that rs3743591G allele is a susceptibility factor for the onset of children with HLH (OR=2.171,95% CI=1.308-3.603) the case group. The frequency of rs2017662AA genotype was higher than the control group, the difference was statistically significant (11% vs.1.4%, P=0.001), suggesting that rs2017662AA genotype was the incidence of pediatric HLH predisposing factors (OR=8.899,95% CI=1.928-41.082); rs2017662A allele frequency in case group was higher than the control group, the difference was statistically significant (21% vs.10.3%, P=0.001, rs2017662A) allele is a susceptibility factor for the onset of children with HLH (OR=2.322,95% 0=1.396-3.860). (3) CX3CR1 gene polymorphism analysis results: the case group rs2853 The frequency of 712TT genotype was higher than the control group, the difference was statistically significant (72% vs.50.0%, P=0.001), suggesting that rs2853712TT genotype is susceptible children factor for the onset of HLH (OR=2.571,95% CI=1.493-4.430). (4) CCR2 gene polymorphism analysis results: the case group rs1799865TT genotype frequency was higher than the control group, the difference was statistically significant (vs.20.5% 39%. P=0.002), suggesting that rs1799865TT genotype is susceptible children factor for the onset of HLH (OR=2.472,95% CI=1.401-4.363); group rS 1799865T allele frequency was higher than the control group, the difference was statistically significant (53% vs.37.0%, P=0.000), suggesting that rs1799865T allele is a susceptibility factor for the onset of children with HLH (OR=1.921,95% CI=1.333-2.769) (5). The results of haplotype analysis: haplotype A-T-G-G-G-C-T-T-T-T-T-A-G-A-C (SNPs sequence: rs3743591-rs11570151-rs2017662-rs2071336-rs2669850-rs17793056-rs13088991-rs 13062158-rs2853712-rs2669841-rs2853711-rs3762823-rs309296 3-rs3092962-rsl 799865) in case group frequency was higher than the control group, the difference was statistically significant (5.7% vs.1.3%, P=0.003), suggesting that haplotype A-T-G-G-G-C-T-T-T-T-T-A-G-A-C is a susceptible factor for the onset of HLH (OR=5.025,95% CI=1.530-16.505). The haplotype G-T-A-G-G-T-C-C-T-C-G-A-G-A-C (SNPsJl sequence: rs3743591-rs11570151-rs2017662-rs2071336-rs2669850-rs17793056-rs13088991-rs13062158-rs28537 12-rs2669841-rs2853711-rs3762823-rs3092963-rs3092962-rs 1799865) in case group frequency was higher than the control group, there are statistically significant difference (5.2%VS.0%, P=4.43 * 10-5), suggesting that haplotype G-T-A-G-G-T-C-C-T-C-G-A-G-A-C is the predisposing factors in the pathogenesis of HLH in children. Children with HLH (6) cases, carrying rs3743591GG genotype had a median survival time of less than carrying rs374359 The 1AA/AG genotype, the difference was statistically significant (86 months vs.118.0 months, P=0.009) carrying the rs2017662AA genotype; the median survival time of less than rs2017662GG/GA genotype, the difference was statistically significant (88 months vs.118.0 months, P=0.004); rs2853712CC/CT genotype rs2853712TT genotype and the average survival time of the patients with who have no significant difference (96.5 months vS.108.6 months, P=0.124) carrying the rs1799865TT genotype; the median survival time of less than rs1799865CC/CT genotype, the difference was statistically significant (92 months vs.107.0 months, P=0.021). Conclusion (1) the research and application of bioinformatics and identified 45 genes the new children's HLH candidate disease, including 42 up-regulated genes and 3 downregulated genes. (2) TNFRSF17, CX3CR1 and CCR2 gene expression in children with HLH, suggesting that it may be involved in The pathogenesis of HLH in children. (3) TNFRSF17 gene rs3743591, rs2017662, CX3CR1, rs2853712 gene, CCR2 gene rs1799865 polymorphism and the pathogenesis of HLH in children. (4) TNFRSF17 gene, CX3CR1 gene and CCR2 gene polymorphism of haplotype A-T-G-G-G-C-T-T-T-T-T-A-G-A-C and G-T-A-G-G-T-C-C-T-C-G-A-G-A-C in children with HLH related disease. (5) TNFRSF17 gene rs3743591, rs2017662 CCR2, rs1799865 gene polymorphisms may serve as prognostic markers in children with HLH.A-T-G-G-G-C-T-T-T-T-T-A-G-A-C and G-T-A-G-G-T-C-C-T-C-G-A-G-A-C in children with HLH related disease. The second part children with hemophagocytic lymphohistiocytosis SH2D1A, XIAP gene mutation and clinical significance to understand the SH2 (SH2 domain protein 1A 1A protein, SH2D1A) gene and X linked inhibitor of apoptosis protein (X-linked inhibitor of apoptosis protein, XIAP) gene The occurrence of mutations and sequence variation in the HLH children of SH2D1A, relationship between XIAP gene mutation and HLH in children. Methods the case group included 100 cases of HLH patients, the control group consisted of 100 healthy children. Peripheral venous blood, genomic DNA was extracted using PCR amplified 1-4 exons of SH2D1A gene and XIAP gene the 1-6 exon encoding region and flanking intron fragment of PCR products by ABI PRISM3730 automatic DNA sequencing direct sequencing, and sequence with SH2D1A and XIAP NCBI gene database for comparison, to obtain genetic variation information, using bioinformatics methods to predict SH2D1A and effect of XIAP mutations on protein function were reviewed. Analysis of the clinical data of the cases of gene mutation, clinical phenotype and genotype were analyzed. Results (1) XIAP gene was found in 1 new missense mutations, 30544 TA HLH in the case group, located in exon first On the change result in amino acid encoding, the Phe27Ile. mutation was not found in the control group, the incidence of mutation was 1% (1/100). (2) predicted XIAP gene missense mutation function using PolyPhen2 and SIFT software, XIAP Phe27Ile gene mutation may affect protein function. (3) found 5 the SNPs sites in the case group and the control group of the Communist Party of China, including the SH2D1A gene rs72610640 rs182286559 polymorphism and XIAP gene of rs5956583, rs140997240, rs28382740 polymorphism; in the case group and the control group compared the genotype of 5 SNPs loci, allele frequency distributions, differences were not statistically significant, suggesting that these SNPs loci may be associated with the pathogenesis of childhood HLH. (4) clinical analysis: 1 cases of children with Phe27Ile mutations had no obvious HLH positive family history and special history, 13 years of onset, clinical manifestations of children with HLH, accept according to HLH-2004. Chemo was alleviated, but recurrence after discontinuation of treatment, the effect is poor, poor prognosis, died of multiple organ failure at the age of 16 (Multiple system organ failure, MSOF). Conclusion this study found 1 novel XIAP missense mutation, the Phe27Ile mutation may be associated with the occurrence and prognosis of children with HLH.

【学位授予单位】：广西医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R725.5
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本文编号：1564866