高通量测序分析肝移植急性排斥反应相关基因单核苷酸多态性位点的突变
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摘要
目的利用高通量测序分析肝移植术后急性排斥反应相关度最高的基因单核苷酸多态性(SNP)位点的突变情况。方法收集同种异体原位肝移植术的68例受体外周血样本,根据有否发生急性排斥反应分为急性排斥组(13例)和非排斥组(55例)。通过查阅文献,最终确定与排斥反应发生相关的44个SNP突变位点。以44个SNP位点作为检测靶点,用高通量测序分析对两组受体外周血样本进行检测,经生物信息学分析出与急性排斥反应发生相关基因SNP位点的突变率。结果急性排斥组中的白细胞介素(IL)-10 TT基因型、T等位基因、AA基因型、A等位基因的SNP位点突变率明显高于非排斥组;急性排斥组中的细胞趋化因子受体(CCR)5AG基因型的SNP位点突变率明显低于非排斥组,CCR5 GG基因型的SNP位点突变率明显高于非排斥组;急性排斥组中的IL-4 CT基因型的SNP位点突变率明显高于非排斥组,IL-4 TT基因型的SNP位点突变率明显低于非排斥组;急性排斥组中核因子-κB抑制因子α(NF-κBIA)C等位基因的SNP位点突变率明显高于非排斥组;急性排斥组中维生素D受体(VDR)CC基因型和C等位基因的SNP位点突变率明显低于非排斥组,差异均有统计学意义(均为P<0.05)。结论高通量测序分析发现肝移植术后急性排斥反应相关基因中,其SNP位点突变率较高的包括IL-10 TT基因型、T等位基因、AA基因型、A等位基因,CCR5 GG基因型、AG基因型,IL-4 CT基因型、TT基因型,NF-κBIA C等位基因,VDR CC基因型和C等位基因。
Objective To analyze the mutation of single nucleotide polymorphism(SNP) loci in genes most significantly associated with acute rejection after liver transplantation by high-throughput sequencing. Methods Peripheral blood samples were collected from 68 recipients undergoing allogeneic orthotopic liver transplantation. According to the incidence of acute rejection, all patients were divided into the acute rejection group(n=13) and non-rejection group(n=55).Through the literature review, 44 mutant SNP loci associated with acute rejection were finally identified. Using 44 SNP loci as the detection targets, high-throughput sequencing analysis was performed to detect peripheral blood samples in two groups of recipients. Bioinformatics analysis revealed the mutation rate of the SNP loci of genes related to acute rejection.Results The mutation rate of SNP loci of the interleukin(IL)-10 TT genotype, T allele, AA genotype and A allele in the acute rejection group was significantly higher than that in the non-rejection group. The mutation rate of the SNP loci of the cell chemokine receptor(CCR) 5 AG genotype in the acute rejection group was significantly lower than that in the nonrejection group, the mutation rate of the SNP loci of the CCR5 GG genotype in the acute rejection group was significantly higher than that in the non-rejection group. The mutation rate of the SNP loci of IL-4 CT genotype in the acute rejection group was significantly higher than that in the non-rejection group, the mutation rate of the SNP loci of IL-4 TT genotype in the acute rejection group was significantly lower than that in the non-rejection group. The mutation rate of the SNP loci of nuclear factor-kappa B inhibitor alpha(NF-κBIA) C allele in the acute rejection group was significantly higher than that in the non-rejection group. The mutation rate of the SNP loci of vitamin D receptor(VDR) CC genotype and C allele in the acute rejection group was significantly lower than that in the non-rejection group. Differences were statistically significant(all P<0.05). Conclusions High-throughput sequencing analysis shows the genes associated with acute rejection after liver transplantation. Among them, the mutation rate of the SNP loci is relatively high in IL-10 TT genotype, T allele,AA genotype, A allele, CCR5 GG genotype, AG genotype, IL-4 CT genotype, TT genotype, NF-κBIA C allele, VDR CC genotype and C allele.
Objective To analyze the mutation of single nucleotide polymorphism(SNP) loci in genes most significantly associated with acute rejection after liver transplantation by high-throughput sequencing. Methods Peripheral blood samples were collected from 68 recipients undergoing allogeneic orthotopic liver transplantation. According to the incidence of acute rejection, all patients were divided into the acute rejection group(n=13) and non-rejection group(n=55).Through the literature review, 44 mutant SNP loci associated with acute rejection were finally identified. Using 44 SNP loci as the detection targets, high-throughput sequencing analysis was performed to detect peripheral blood samples in two groups of recipients. Bioinformatics analysis revealed the mutation rate of the SNP loci of genes related to acute rejection.Results The mutation rate of SNP loci of the interleukin(IL)-10 TT genotype, T allele, AA genotype and A allele in the acute rejection group was significantly higher than that in the non-rejection group. The mutation rate of the SNP loci of the cell chemokine receptor(CCR) 5 AG genotype in the acute rejection group was significantly lower than that in the nonrejection group, the mutation rate of the SNP loci of the CCR5 GG genotype in the acute rejection group was significantly higher than that in the non-rejection group. The mutation rate of the SNP loci of IL-4 CT genotype in the acute rejection group was significantly higher than that in the non-rejection group, the mutation rate of the SNP loci of IL-4 TT genotype in the acute rejection group was significantly lower than that in the non-rejection group. The mutation rate of the SNP loci of nuclear factor-kappa B inhibitor alpha(NF-κBIA) C allele in the acute rejection group was significantly higher than that in the non-rejection group. The mutation rate of the SNP loci of vitamin D receptor(VDR) CC genotype and C allele in the acute rejection group was significantly lower than that in the non-rejection group. Differences were statistically significant(all P<0.05). Conclusions High-throughput sequencing analysis shows the genes associated with acute rejection after liver transplantation. Among them, the mutation rate of the SNP loci is relatively high in IL-10 TT genotype, T allele,AA genotype, A allele, CCR5 GG genotype, AG genotype, IL-4 CT genotype, TT genotype, NF-κBIA C allele, VDR CC genotype and C allele.
引文
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[17]FALLETI E,BITETTO D,FABRIS C,et al.Association between vitamin D receptor genetic polymorphisms and acute cellular rejection in liver-transplanted patients[J].Transpl Int,2012,25(3):314-322.DOI:10.1111/j.1432-2277.2011.01419.x.
[2]徐翠香,靳占奎,王曦,等.Luminex技术检测肾移植患者血清IL-17预测急性排斥反应的回顾性研究[J].现代检验医学杂志,2017,32(4):87-90.DOI:10.3969/j.issn.1671-7414.2017.04.024.XU CX,JIN ZK,WANG X,et al.Retrospective study of detection of serum IL-17 for predicting early acute renal allograft rejection by Luminex technique[J].J Mod Lab Med,2017,32(4):87-90.DOI:10.3969/j.issn.1671-7414.2017.04.024.
[3]VERHELST XP,TROISI RI,COLLE I,et al.Biomarkers for the diagnosis of acute cellular rejection in liver transplant recipients:a review[J].Hepatol Res,2013,43(2):165-178.DOI:10.1111/hepr.12012.
[4]杨扬,邓宜南.抗体免疫诱导治疗在肝移植中的应用[J].临床肝胆病杂志,2015,31(12):2031-2034.DOI:10.3969/j.issn.1001-5256.2015.12.010.YANG Y,DENG YN.Application of immune induction therapy with antibodies in liver transplantation[J].J Clin Hepatol,2015,31(12):2031-2034.DOI:10.3969/j.issn.1001-5256.2015.12.010.
[5]AGUADO-DOMíNGUEZ E,GóMEZ L,SOUSA JM,et al.Identification of the cellular components involved in de novo immune hepatitis:a quantitative immunohistochemical analysis[J].J Transl Med,2018,16(1):62.DOI:10.1186/s12967-018-1440-8.
[6]MORI S D,TS ILIMIGRAS DI,NTANASISSTATHOPOULOS I,et al.Liver transplantation in patients with liver metastases from neuroendocrine tumors:a systematic review[J].Surgery,2017,162(3):525-536.DOI:10.1016/j.surg.2017.05.006.
[7]RODRíGUEZ-PERáLVAREZ M,GERMANI G,TSOCHATZIS E,et al.Predicting severity and clinical course of acute rejection after liver transplantation using blood eosinophil count[J].Transpl Int,2012,25(5):555-563.DOI:10.1111/j.1432-2277.2012.01457.x.
[8]LIU XQ,HU ZQ,PEI YF,et al.Clinical operational tolerance in liver transplantation:state-of-the-art perspective and future prospects[J].Hepatobiliary Pancreat Dis Int,2013,12(1):12-33.
[9]CHEN Y,ZHANG H,XIAO X,et al.Peripheral blood transcriptome sequencing reveals rejection-relevant genes in long-term heart transplantation[J].Int J Cardiol,2013,168(3):2726-2733.DOI:10.1016/j.ijcard.2013.03.095.
[10]WHEELER DA,SRINIVASAN M,EGHOLM M,et al.The complete genome of an individual by massively parallel DNA sequencing[J].Nature,2008,452(7189):872-876.DOI:10.1038/nature06884.
[11]REUTER JA,SPACEK DV,SNYDER MP.Highthroughput sequencing technologies[J].Mol Cell,2015,58(4):586-597.DOI:10.1016/j.molcel.2015.05.004.
[12]TURNER DM,WILLIAMS DM,SANKARAN D,et al.An investigation of polymorphism in the interleukin-10gene promoter[J].Eur J Immunogenet,1997,24(1):1-8.
[13]苏青和,郑红.趋化因子及树突状细胞与移植免疫耐受[J].免疫学杂志,2004,20(5):403-406.DOI:10.3969/j.issn.1000-8861.2004.05.021.SU QH,ZHENG H.Dendritic cells,chemokines,and graft immune tolerance[J].Immunol J,2004,20(5):403-406.DOI:10.3969/j.issn.1000-8861.2004.05.021.
[14]杨蕾,刘永锋,王凤山,等.趋化因子受体多态性与肝移植急性排斥反应的关系[J].免疫学杂志,2008,24(2):249.DOI:10.3969/j.issn.1000-8861.2008.02.035.YANG L,LIU YF,WANG FS,et al.Association between polymorphism of chemokine receptor and acute rejection in liver transplantation[J].Immunol J,2008,24(2):249.DOI:10.3969/j.issn.1000-8861.2008.02.035.
[15]WU W,LIU Y,LI S,et al.Association between IL-4polymorphism and acute rejection of solid organ allograft:a Meta-analysis[J].Gene,2013,513(1):14-21.DOI:10.1016/j.gene.2012.10.028.
[16]KRAMER K,THYE T,TRESZL A,et al.Polymorphism in NFκBIA gene is associated with recurrent acute rejections in liver transplant recipients[J].Tissue Antigens,2014,84(4):370-377.DOI:10.1111/tan.12411.
[17]FALLETI E,BITETTO D,FABRIS C,et al.Association between vitamin D receptor genetic polymorphisms and acute cellular rejection in liver-transplanted patients[J].Transpl Int,2012,25(3):314-322.DOI:10.1111/j.1432-2277.2011.01419.x.