發(fā)布時(shí)間：2018-03-04 08:18

  本文選題：噬血性淋巴組織細(xì)胞增生癥　切入點(diǎn)：兒童　出處：《廣西醫(yī)科大學(xué)》2015年博士論文　論文類(lèi)型：學(xué)位論文

【摘要】：第一部分基于基因表達(dá)譜的兒童噬血性淋巴組織細(xì)胞增生癥候選基因篩選及相關(guān)實(shí)驗(yàn)研究目的應(yīng)用生物信息學(xué)方法,篩選新的兒童噬血性淋巴組織細(xì)胞增生癥(He-mophagocytic lymphohistiocytosis, HLH)候選疾病基因并進(jìn)行實(shí)驗(yàn)驗(yàn)證,探討兒童HLH發(fā)病和預(yù)后的分子機(jī)制。方法(1)從基因表達(dá)綜合數(shù)據(jù)庫(kù)(Gene expression omnibus, GEO)中下載GSE26050兒童HLH基因芯片數(shù)據(jù)集,然后在R語(yǔ)言軟件包中進(jìn)行差異表達(dá)基因(Differentially expressed genes, DEGs)分析,將獲得的DEGs定義為“檢測(cè)基因集”；采用Genecards和Fable文獻(xiàn)挖掘已知兒童HLH疾病基因,將其定義為“訓(xùn)練基因集”；最后,利用Toppgene在線(xiàn)分析工具篩選出兒童HLH候選疾病基因。(2)以100例HLH患兒為病例組,146例健康兒童為對(duì)照組,采集外周靜脈血,采用多重聚合酶鏈?zhǔn)椒磻?yīng)(Polymerase chain reaction, PCR)和SNaPshot技術(shù)對(duì)部分候選基因單核苷酸多態(tài)性(Single nucleotide polymorphism, SNP)進(jìn)行檢測(cè)。(3)用Haploview v.4.2軟件進(jìn)行各SNP位點(diǎn)哈-溫平衡(Hardy-Weinberg eq-uilibrium, HWE)和相互間的連鎖不平衡(Linkage disequilibrium, LD)檢驗(yàn)。用SPSS17.0軟件分析各位點(diǎn)基因型頻率、等位基因頻率分布差異與兒童HLH發(fā)病的相關(guān)性。風(fēng)險(xiǎn)度采用比值比(Odds ratio, OR)及其95%可信區(qū)間(Confidence interval,CI)表示。用PHASE2.1軟件進(jìn)行單體型的構(gòu)建和分析。P小于0.05視為差異有統(tǒng)計(jì)學(xué)意義。(4)對(duì)入選HLH患兒進(jìn)行病歷資料收集并隨訪(fǎng)。以確診HLH為觀(guān)察起點(diǎn),死亡日期作為觀(guān)察終點(diǎn),失訪(fǎng)、末次隨訪(fǎng)時(shí)仍存活(包括緩解、疾病活動(dòng)、疾病復(fù)發(fā)的患兒)的病例則確定為截尾病例。以有意義的SNPs不同基因型作為研究因素,采用Kaplan-Meier曲線(xiàn)進(jìn)行生存分析,應(yīng)用Log-rank檢驗(yàn)比較生存曲線(xiàn)。以P0.05為差異有統(tǒng)計(jì)學(xué)意義。結(jié)果(1)獲得一個(gè)含86個(gè)基因的“檢測(cè)基因集”和一個(gè)含73個(gè)基因的“訓(xùn)練基因集”；采用Toppgene共獲得45個(gè)兒童HLH候選疾病基因,其中表達(dá)上調(diào)的基因42個(gè),表達(dá)下調(diào)的基因3個(gè),為T(mén)NFRSF17、CX3CR1和CCR2。(2)TNFRSF17基因多態(tài)性分析結(jié)果：病例組rs3743591GG基因型頻率高于對(duì)照組,差異有統(tǒng)計(jì)學(xué)意義(10.0% vs.1.4%,P=0.002),提示rs3743591GG基因型是兒童HLH發(fā)病的易感因素(OR=8.000,95% CI=1.714-37.349);病例組rs3743591G等位基因頻率高于對(duì)照組,差異有統(tǒng)計(jì)學(xué)意義(20.5% vs.10.6%,P=0.002),提示rs3743591G等位基因是兒童HLH發(fā)病的易感因素(OR=2.171,95% CI=1.308-3.603)。病例組rs2017662AA基因型頻率高于對(duì)照組,差異有統(tǒng)計(jì)學(xué)意義(11.0% vs.1.4%,P=0.001),提示rs2017662AA基因型是兒童HLH發(fā)病的易感因素(OR=8.899,95% CI=1.928-41.082);病例組rs2017662A等位基因頻率高于對(duì)照組,差異有統(tǒng)計(jì)學(xué)意義(21.0% vs.10.3%,P=0.001),提示rs2017662A等位基因是兒童HLH發(fā)病的易感因素(OR=2.322,95% 0=1.396-3.860)。(3) CX3CR1基因多態(tài)性分析結(jié)果：病例組rs2853712TT基因型頻率高于對(duì)照組,差異有統(tǒng)計(jì)學(xué)意義(72.0% vs.50.0%,P=0.001),提示rs2853712TT基因型是兒童HLH發(fā)病的易感因素(OR=2.571,95% CI=1.493-4.430)。(4)CCR2基因多態(tài)性分析結(jié)果：病例組rs1799865TT基因型頻率高于對(duì)照組,差異有統(tǒng)計(jì)學(xué)意義(39.0% vs.20.5%,P=0.002),提示rs1799865TT基因型是兒童HLH發(fā)病的易感因素(OR=2.472,95% CI=1.401-4.363);病例組rS 1799865T等位基因頻率高于對(duì)照組,差異有統(tǒng)計(jì)學(xué)意義(53.0% vs.37.0%,P=0.000),提示rs1799865T等位基因是兒童HLH發(fā)病的易感因素(OR=1.921,95% CI=1.333-2.769)。(5)單體型分析結(jié)果：?jiǎn)误w型A-T-G-G-G-C-T-T-T-T-T-A-G-A-C (SNPs順序：rs3743591-rs11570151-rs2017662-rs2071336-rs2669850-rs17793056-rs13088991-rs13062158-rs2853712-rs2669841-rs2853711-rs3762823-rs309296 3-rs3092962-rsl 799865)在病例組出現(xiàn)的頻率高于對(duì)照組,差異有統(tǒng)計(jì)學(xué)意義(5.7% vs.1.3%,P=0.003),提示單體型A-T-G-G-G-C-T-T-T-T-T-A-G-A-C是兒童HLH發(fā)病的易感因素(OR=5.025,95% CI=1.530-16.505).單體型G-T-A-G-G-T-C-C-T-C-G-A-G-A-C (SNPsJl順序：rs3743591-rs11570151-rs2017662-rs2071336-rs2669850-rs17793056-rs13088991-rs13062158-rs28537 12-rs2669841-rs2853711-rs3762823-rs3092963-rs3092962-rs 1799865)在病例組出現(xiàn)的頻率高于對(duì)照組,差異有統(tǒng)計(jì)學(xué)意義(5.2%VS.0%,P=4.43×10-5),提示單體型G-T-A-G-G-T-C-C-T-C-G-A-G-A-C是兒童HLH發(fā)病的易感因素。(6)兒童HLH病例中,攜帶rs3743591GG基因型者中位生存時(shí)間小于攜帶rs3743591AA/AG基因型者,差異有統(tǒng)計(jì)學(xué)意義(86.0個(gè)月vs.118.0個(gè)月,P=0.009);攜帶rs2017662AA基因型者中位生存時(shí)間小于攜帶rs2017662GG/GA基因型者,差異有統(tǒng)計(jì)學(xué)意義(88.0個(gè)月vs.118.0個(gè)月,P=0.004)；攜帶rs2853712TT基因型者平均生存時(shí)間與攜帶rs2853712CC/CT基因型者比較差別無(wú)統(tǒng)計(jì)學(xué)意義(96.5個(gè)月vS.108.6個(gè)月,P=0.124)；攜帶rs1799865TT基因型者中位生存時(shí)間小于攜帶rs1799865CC/CT基因型者,差異有統(tǒng)計(jì)學(xué)意義(92.0個(gè)月vs.107.0個(gè)月,P=0.021)。結(jié)論(1)本研究應(yīng)用生物信息學(xué)方法篩選出45個(gè)新的兒童HLH候選疾病基因,包括42個(gè)上調(diào)表達(dá)基因和3個(gè)下調(diào)表達(dá)基因。(2) TNFRSF17、CX3CR1和CCR2基因在兒童HLH中表達(dá)下調(diào),提示其可能參與兒童HLH的發(fā)病機(jī)制。(3)TNFRSF17基因rs3743591、rs2017662, CX3CR1基因rs2853712, CCR2基因rs1799865多態(tài)位點(diǎn)與兒童HLH發(fā)病有關(guān)。(4) TNFRSF17基因、CX3CR1基因和CCR2基因多態(tài)位點(diǎn)構(gòu)成的單體型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與兒童HLH發(fā)病有關(guān)。(5) TNFRSF17基因rs3743591、rs2017662, CCR2基因rs1799865多態(tài)位點(diǎn)有可能作為兒童HLH預(yù)后判斷的分子標(biāo)記。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與兒童HLH發(fā)病有關(guān)。第二部分兒童噬血性淋巴組織細(xì)胞增生癥SH2D1A、XIAP基因變異和臨床意義目的了解SH2區(qū)蛋白1A (SH2 domain protein 1A, SH2D1A)基因和X連鎖凋亡抑制蛋白(X-linked inhibitor of apoptosis protein, XIAP)基因突變和序列變異在HLH患兒中的發(fā)生情況,探討SH2D1A、XIAP基因變異與兒童HLH的關(guān)系。方法病例組為100例HLH患兒,對(duì)照組為100例健康兒童。采集外周靜脈血,提取基因組DNA,采用PCR法擴(kuò)增SH2D1A基因1-4外顯子和XIAP基因1-6外顯子編碼區(qū)及其側(cè)翼內(nèi)含子區(qū)片段,PCR產(chǎn)物經(jīng)ABI PRISM3730全自動(dòng)DNA測(cè)序儀直接測(cè)序,然后與NCBI數(shù)據(jù)庫(kù)中SH2D1A和XIAP基因的序列進(jìn)行比對(duì),獲得基因變異信息,利用生物信息學(xué)方法預(yù)測(cè)SH2D1A和XIAP基因突變對(duì)蛋白質(zhì)功能的影響。對(duì)基因突變的病例回顧性分析其臨床資料,將其臨床表型與基因型進(jìn)行對(duì)比分析。結(jié)果(1)在HLH病例組中發(fā)現(xiàn)XIAP基因1個(gè)新的錯(cuò)義突變,30544 TA,位于第1外顯子上,導(dǎo)致所編碼的氨基酸發(fā)生改變,Phe27Ile。該位點(diǎn)突變?cè)趯?duì)照組中未發(fā)現(xiàn),突變發(fā)生率為1%(1/100)。(2)用PolyPhen2和SIFT軟件預(yù)測(cè)XIAP基因錯(cuò)義突變功能,顯示XIAP基因Phe27Ile突變有可能影響蛋白質(zhì)功能。(3)在病例組和對(duì)照組中共發(fā)現(xiàn)5個(gè)SNPs位點(diǎn),包括SH2D1A基因的rs72610640、rs182286559多態(tài)和XIAP基因的rs5956583、rs140997240、rs28382740多態(tài)；在病例組和對(duì)照組中比較了這5個(gè)SNPs位點(diǎn)的基因型、等位基因的頻率分布,差異均無(wú)統(tǒng)計(jì)學(xué)意義,提示這些SNPs位點(diǎn)可能與兒童HLH發(fā)病無(wú)關(guān)。(4)臨床特征分析：發(fā)生Phe27Ile突變的1例患兒無(wú)明顯HLH陽(yáng)性家族史和特殊既往史,13歲起病,患兒臨床表現(xiàn)為HLH,按照HLH-2004方案接受化療曾經(jīng)得到緩解,但停藥后復(fù)發(fā),治療效果差,預(yù)后不良,于16歲時(shí)死于多器官功能衰竭(Multiple system organ failure, MSOF)。結(jié)論本研究發(fā)現(xiàn)了1個(gè)新的XIAP基因錯(cuò)義突變,Phe27Ile,該突變可能與HLH患兒的發(fā)生和不良預(yù)后有關(guān)。
[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.

【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R725.5
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本文編號(hào)：1564866