抗结核药物性肝损伤患者外周血基因组DNA甲基化差异的筛查和验证
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摘要
目的采用基因甲基化芯片技术筛选抗结核药物性肝损伤(anti-tuberculosis drug induced liver injury,ADLI)患者与非肝损伤患者间DNA差异甲基化基因位点。探讨DNA甲基化与ADLI发病机制的关系。方法选取2016年1月至2017年7月间唐山市第四医院治疗的肺结核初治患者共137例,分为ADLI组(n=65)、非ADLI组(n=72)。收集研究对象的临床一般资料及全血标本。选择其中14例(7例ADLI,7例非ADLI)患者,提取全血标本的DNA,利用Illumina 850K芯片技术检测病例组和对照组DNA的差异甲基化情况。对芯片筛选出差异较大的甲基化基因DUSP22、HLA-C,进一步在剩余的65例病例组和58例对照组中采用甲基化特异PCR(MSP)方法验证甲基化水平,并采用荧光定量PCR检测DUSP22、HLA-C基因的m RNA水平。结果共检测853 307个CpG位点,病例组和对照组差异显著的位点共1 012个,其中高甲基化位点254个,低甲基化位点758个,主要位于基因体区。聚类分析结果显示,在细胞黏附、金属离子结合等过程中存在大量甲基化差异基因。病例组与对照组人类双特异性磷酸酶DUSP22基因、人类白细胞抗原HLA-C基因的甲基化率差异均有统计学意义(65. 6%vs 36. 9%、37. 9%vs 58. 5%,P <0. 05),对照组中DUSP22 m RNA表达水平高于病例组,而HLA-C m RNA表达水平低于病例组。结论外周血中DUSP22、HLA-C基因DNA甲基化与ADLI的发生相关。
Objective To investigate the differentially methylated genes between patients with antituberculosis drug induced liver injury( ADLI) and patients without hepatic injury using DNA methylation microarrays and explore the relationship between DNA methylation and ADLI. Methods This study was conducted among 137 patients with newly diagnosed pulmonary tuberculosis( including 65 patients with ADLI and 72 without ADLI) treated in the Fourth Hospital of Tangshan City between January,2016 and July,2017. The clinical data were collected and whole blood samples were obtained from all the patients,and 14 patients,including 7 with ADLI and 7 without ADLI,were selected for examination of DNA methylation status using Illumina 850 K bead-chip. The methylation status of the 2 differentially methylated genes,DUSP22 and HLA-C,which were identified by analysis of the microarray data,were verified in the remaining cases using methylation-specific PCR( MSP); RT-PCR was used to detect the m RNA levels of DUSP22 and HLA-C genes in all the patients. Results We analyzed a total of 853 307 loci and identified 1 012 differential methylated loci between ADLI group and non-ADLI group,including 254 hypermethylated and 758 hypomethylated loci,located mainly in the gene body regions. Cluster analysis showed that a large number of differentially methylated genes were involved in the process of cell adhesion and metal ion binding. MSP verified that the methylation rates of DUSP22 and HLA-C genes differed significantly between ADLI group and non-ADLI group( 65. 6% vs 36. 9%; 37. 9% vs 58. 5%; both P < 0. 05). The m RNA expression of DUSP22 was higher and that of HLA-C was lower in non-ADLI group than in ADLI group. Conclusion DNA methylation of DUSP22 and HLA-C in the peripheral blood is associated with the development of ADLI.
Objective To investigate the differentially methylated genes between patients with antituberculosis drug induced liver injury( ADLI) and patients without hepatic injury using DNA methylation microarrays and explore the relationship between DNA methylation and ADLI. Methods This study was conducted among 137 patients with newly diagnosed pulmonary tuberculosis( including 65 patients with ADLI and 72 without ADLI) treated in the Fourth Hospital of Tangshan City between January,2016 and July,2017. The clinical data were collected and whole blood samples were obtained from all the patients,and 14 patients,including 7 with ADLI and 7 without ADLI,were selected for examination of DNA methylation status using Illumina 850 K bead-chip. The methylation status of the 2 differentially methylated genes,DUSP22 and HLA-C,which were identified by analysis of the microarray data,were verified in the remaining cases using methylation-specific PCR( MSP); RT-PCR was used to detect the m RNA levels of DUSP22 and HLA-C genes in all the patients. Results We analyzed a total of 853 307 loci and identified 1 012 differential methylated loci between ADLI group and non-ADLI group,including 254 hypermethylated and 758 hypomethylated loci,located mainly in the gene body regions. Cluster analysis showed that a large number of differentially methylated genes were involved in the process of cell adhesion and metal ion binding. MSP verified that the methylation rates of DUSP22 and HLA-C genes differed significantly between ADLI group and non-ADLI group( 65. 6% vs 36. 9%; 37. 9% vs 58. 5%; both P < 0. 05). The m RNA expression of DUSP22 was higher and that of HLA-C was lower in non-ADLI group than in ADLI group. Conclusion DNA methylation of DUSP22 and HLA-C in the peripheral blood is associated with the development of ADLI.
引文
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[11] RAUSCHER G H,KRESOVICH J K,POULIN M,et al.Exploring DNA methylation changes in promoter,intragenic,and intergenic regions as early and late events in breast cancer formation[J]. BMC Cancer,2015,15(1). DOI:10. 1186/s12885-015-1777-9.
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[13] XIE W,BARR C L,KIM A,et al. Base-resolution analyses of sequence and parent-of-origin dependent DNA methylation in the mouse genome[J]. Cell,2012,148(4):816-831.DOI:10. 1016/j. cell. 2011. 12. 035.
[14] YANG X,HAN H,DE CARVALHO D D,et al. Gene body methylation can alter gene expression and is a therapeutic target in cancer[J]. Cancer Cell,2014,26(4):577-590. DOI:10. 1016/j. ccr. 2014. 07. 028.
[15] HUANG C Y,TAN T H. DUSPs,to MAP kinases and beyond[J]. Cell Biosci,2012,2(1):24. DOI:10. 1186/2045-3701-2-24.
[16] JU A,CHO Y C,KIM B R,et al. Scaffold role of DUSP22in ASK1-MKK7-JNK signaling pathway[J]. PLo S ONE,2016,11(10):e0164259. DOI:10. 1371/journal. pone.0164259.
[17] SANCHEZ-MUT J V,ASO E,HEYN H,et al. Promoter hypermethylation of the phosphatase DUSP22 mediates PKAdependent TAU phosphorylation and CREB activation in alzheimer’s disease[J]. Hippocampus,2014,24(4):363-368. DOI:10. 1002/hipo. 22245.
[18]王书杰,王沛,李晓天,等.物性肝损伤的代谢、遗传学机制[J].中国药理学通报,2016,32(7):898-902.DOI:10. 3969/j. issn. 1001-1978. 2016. 07. 003.WANG S J,WANG P,LI X T,et al. Drug-induced liver injury:metabolic and genetic basis[J]. Chin Pharmacol Bull,2016,32(7):898-902. DOI:10. 3969/j. issn.1001-1978. 2016. 07. 003.
[2] RUSSMANN S,JETTER A,KULLAK-UBLICK G A. Pharmacogenetics of drug-induced liver injury[J]. Hepatology,2010,52(2):748-761. DOI:10. 1002/hep. 23720.
[3] KUMAR S,CHENG X,KLIMASAUSKAS S,et al. The DNA(cytosine-5)methyltransferases[J]. Nucleic Acids Res,1994,22(1):1-10.
[4]中华医学会结核病学分会.肺结核诊断和治疗指南[J].中华结核和呼吸杂志,2001,24(2):70-74.TB Society of Chinese Medical Association. Guidelines for diagnosis and treatment of tuberculosis?[J]. Chin J Tuber Respir Dis,2001,24(2):70-74.
[5]许绍发,端木宏谨.肺外结核的诊断和治疗[J].中华结核和呼吸杂志,1997,20(3):131-132.XU S F,DUANMU H J. Diagnosis and treatment of extrapulmonary tuberculosis[J]. Chin J Tuber Respir Dis,1997,20(3):131-132.
[6] BéNICHOU C. Criteria of drug-induced liver disorders.Report of an international consensus meeting[J]. J Hepatol,1990,11(2):272-276.
[7] JELTSCH A,JURKOWSKA R Z. New concepts in DNA methylation[J]. Trends Biochem Sci,2014,39(7):310-318. DOI:10. 1016/j. tibs. 2014. 05. 002.
[8] QU J,HODGES E,MOLARO A,et al. Evolutionary expansion of DNA hypomethylation in the mammalian germline genome[J]. Genome Res,2018,28(2):145-158. DOI:10. 1101/gr. 225896. 117.
[9] NAKAGAWA T,KANAI Y,USHIJIMA S,et al. DNA hypomethylation on pericentromeric satellite regions significantly correlates with loss of heterozygosity on chromosome 9in urothelial carcinomas[J]. J Urol,2005,173(1):243-246. DOI:10. 1097/01. ju. 0000141577. 98902. 49.
[10] VAN TONGELEN A,LORIOT A,DE SMET C. Oncogenic roles of DNA hypomethylation through the activation of cancer-germline genes[J]. Cancer Lett,2017,396:130-137.DOI:10. 1016/j. canlet. 2017. 03. 029.
[11] RAUSCHER G H,KRESOVICH J K,POULIN M,et al.Exploring DNA methylation changes in promoter,intragenic,and intergenic regions as early and late events in breast cancer formation[J]. BMC Cancer,2015,15(1). DOI:10. 1186/s12885-015-1777-9.
[12] ARAN D,TOPEROFF G,ROSENBERG M,et al. Replication timing-related and gene body-specific methylation of active human genes[J]. Hum Mol Genet,2011,20(4):670-680. DOI:10. 1093/hmg/ddq513.
[13] XIE W,BARR C L,KIM A,et al. Base-resolution analyses of sequence and parent-of-origin dependent DNA methylation in the mouse genome[J]. Cell,2012,148(4):816-831.DOI:10. 1016/j. cell. 2011. 12. 035.
[14] YANG X,HAN H,DE CARVALHO D D,et al. Gene body methylation can alter gene expression and is a therapeutic target in cancer[J]. Cancer Cell,2014,26(4):577-590. DOI:10. 1016/j. ccr. 2014. 07. 028.
[15] HUANG C Y,TAN T H. DUSPs,to MAP kinases and beyond[J]. Cell Biosci,2012,2(1):24. DOI:10. 1186/2045-3701-2-24.
[16] JU A,CHO Y C,KIM B R,et al. Scaffold role of DUSP22in ASK1-MKK7-JNK signaling pathway[J]. PLo S ONE,2016,11(10):e0164259. DOI:10. 1371/journal. pone.0164259.
[17] SANCHEZ-MUT J V,ASO E,HEYN H,et al. Promoter hypermethylation of the phosphatase DUSP22 mediates PKAdependent TAU phosphorylation and CREB activation in alzheimer’s disease[J]. Hippocampus,2014,24(4):363-368. DOI:10. 1002/hipo. 22245.
[18]王书杰,王沛,李晓天,等.物性肝损伤的代谢、遗传学机制[J].中国药理学通报,2016,32(7):898-902.DOI:10. 3969/j. issn. 1001-1978. 2016. 07. 003.WANG S J,WANG P,LI X T,et al. Drug-induced liver injury:metabolic and genetic basis[J]. Chin Pharmacol Bull,2016,32(7):898-902. DOI:10. 3969/j. issn.1001-1978. 2016. 07. 003.