系统性红斑狼疮候选基因的多态性及其与疾病的关联研究
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摘要
系统性红斑狼疮(Systemic Lupus Erythematosus，SLE)是一种特异性的自身免疫疾病，被公认为自身免疫疾病的原型。通过对群体遗传学、双生子发病一致率和疾病家族聚集性等一系列研究均证实遗传因素在SLE发病中起着重要作用。
目前对于Toll样受体(Toll-like Receptors，TLRs)的研究揭示该通路系统调节着天然免疫系统与获得性免疫系统之间的平衡，与免疫缺陷性疾病、异常免疫应答性疾病、关节炎等疾病的发病有关。
我们应用了聚合酶链反应(polymerase chain reaction，PCR)、Sanger双脱氧测序法和Pyrosequencing焦磷酸测序法，研究了TLR9信号通路内相关基因TLR9、MYD88、TRAF6和IRF7基因的变异情况，并通过病例对照研究探讨了TRAF6基因单核苷酸多态性与SLE易感性的关系。
我们还建立了凝胶电泳迁移率变动分析(electrophoretic mobility shift assay，EMSA)化学发光法检测外周血单个核细胞细胞核内干扰素调节因子7(interferon regulatory factor 7，IRF7)活性的方法。这为进一步研究TLR9通路最下游信号分子IRF7对于IFN-a基因的转录调控作用奠定了基础。
2’，5’-寡聚核苷酸合成酶(2’，5’-oligoadenylate synthetase，2’5’AS)是比较重要的IFN诱导蛋白家族，参与了机体天然抗病毒免疫应答反应。目前，已发现2’5’AS内成员OAS1基因单核苷酸多态性与自身免疫疾病Ⅰ型糖尿病的易感性相关。
我们采用了家系传递不平衡检验方法，探讨了OAS1基因多态性在SLE家系中的传递规律，以及多态性位点与SLE的相关性研究。
结果发现：
(1) 中国汉族人群中TLR9基因内部存在多态性为rs352140，rs352139和rs187084。MYD88基因内部存在多态性为rs4988457和rs7744。此外在实验中，我们还发现一个新SNP位于MYD88基因5’端调控区。IRF7基因调控功能区及编码区存在多态性为rs2277270和rs1061502。证实了TRAF6基因调控区多态性位点rs540386和rs5030437。
(2) 病例对照研究结果显示：中国汉族人群中TRAF6基因单核苷酸多态性rs540386等位基因(T)频率在SLE患者组(2.4％)和正常对照组(8.7％)中差异存在统计学意义(p=0.002)；TT基因型的频率在SLE患者组(4％)与在正常对照组(14.2％)中相比显著下降
Systemic lupus erythematosus (SLE) is a generally accepted prototype autoimmune disease. The importance of genetic influences on SLE has been consistently supported by studies of populations, twin concordance rates, and aggregation of disease in families.
Recent studies have reported that Toll-like receptors are key regulators of both the initial innate and subsequent adaptive immune response. These studies have shown that TLR signaling affects several diseases, including immunodeficiencies, undesirable immune response and arithritis.
In this study, polymerase chain reaction, Sanger sequencing and Pyrosequencing techniques are applied in investigation of genetic polymorphisms in genes (TLR9, MyD88, TRAF6, IRF7) that participate in TLR9 signaling pathway. Case-control study is carried out to study the contribution of TRAF6 single nucleotide polymorphisms to the risk of SLE in Chinese population.
We establish a method for detection of IRF7 activity in human peripheral blood mononuclear cells using gel electrophoretic mobility shift assay and enhanced chem iluminescent technique, which gives a foundation for further study on how IRF7, downstream of TLR9 pathway, regulates interferon-a expression.
2',5'-oligoadenylate synthetase family is a group of interferon-stimulated genes that play important roles in innate antiviral defence. Recently, the minor allele of an OAS1 SNP that alters splicing was found to be associated with increased enzymatic activity and with type 1 diabetes. In this study, we investigate OAS1 SNP with SLE association in 250 nuclear families using transmission disequilibrium test.
We find:
(1) Three SNPs, rs352130, rs352140 and rsl87084 are identified in TLR9 genomic region. Two SNPs, rs4988457and rs7744 are identified in MyD88 genomic region. Furthermore, a new SNP is found in MyD88 5' regulatory region. rsl061502 and rs2277270 are identified in IRF7 coding region and regulatory region. There are also two SNPs, rs540386 and rs5030437 locate in first intron of TRAF6.
(2) The minor rs540386 T allele frequency was significantly lower in SLE cases than controls (p<0.05). There are 2.4% of SLE cases carrying the
目前对于Toll样受体(Toll-like Receptors，TLRs)的研究揭示该通路系统调节着天然免疫系统与获得性免疫系统之间的平衡，与免疫缺陷性疾病、异常免疫应答性疾病、关节炎等疾病的发病有关。
我们应用了聚合酶链反应(polymerase chain reaction，PCR)、Sanger双脱氧测序法和Pyrosequencing焦磷酸测序法，研究了TLR9信号通路内相关基因TLR9、MYD88、TRAF6和IRF7基因的变异情况，并通过病例对照研究探讨了TRAF6基因单核苷酸多态性与SLE易感性的关系。
我们还建立了凝胶电泳迁移率变动分析(electrophoretic mobility shift assay，EMSA)化学发光法检测外周血单个核细胞细胞核内干扰素调节因子7(interferon regulatory factor 7，IRF7)活性的方法。这为进一步研究TLR9通路最下游信号分子IRF7对于IFN-a基因的转录调控作用奠定了基础。
2’，5’-寡聚核苷酸合成酶(2’，5’-oligoadenylate synthetase，2’5’AS)是比较重要的IFN诱导蛋白家族，参与了机体天然抗病毒免疫应答反应。目前，已发现2’5’AS内成员OAS1基因单核苷酸多态性与自身免疫疾病Ⅰ型糖尿病的易感性相关。
我们采用了家系传递不平衡检验方法，探讨了OAS1基因多态性在SLE家系中的传递规律，以及多态性位点与SLE的相关性研究。
结果发现：
(1) 中国汉族人群中TLR9基因内部存在多态性为rs352140，rs352139和rs187084。MYD88基因内部存在多态性为rs4988457和rs7744。此外在实验中，我们还发现一个新SNP位于MYD88基因5’端调控区。IRF7基因调控功能区及编码区存在多态性为rs2277270和rs1061502。证实了TRAF6基因调控区多态性位点rs540386和rs5030437。
(2) 病例对照研究结果显示：中国汉族人群中TRAF6基因单核苷酸多态性rs540386等位基因(T)频率在SLE患者组(2.4％)和正常对照组(8.7％)中差异存在统计学意义(p=0.002)；TT基因型的频率在SLE患者组(4％)与在正常对照组(14.2％)中相比显著下降
Systemic lupus erythematosus (SLE) is a generally accepted prototype autoimmune disease. The importance of genetic influences on SLE has been consistently supported by studies of populations, twin concordance rates, and aggregation of disease in families.
Recent studies have reported that Toll-like receptors are key regulators of both the initial innate and subsequent adaptive immune response. These studies have shown that TLR signaling affects several diseases, including immunodeficiencies, undesirable immune response and arithritis.
In this study, polymerase chain reaction, Sanger sequencing and Pyrosequencing techniques are applied in investigation of genetic polymorphisms in genes (TLR9, MyD88, TRAF6, IRF7) that participate in TLR9 signaling pathway. Case-control study is carried out to study the contribution of TRAF6 single nucleotide polymorphisms to the risk of SLE in Chinese population.
We establish a method for detection of IRF7 activity in human peripheral blood mononuclear cells using gel electrophoretic mobility shift assay and enhanced chem iluminescent technique, which gives a foundation for further study on how IRF7, downstream of TLR9 pathway, regulates interferon-a expression.
2',5'-oligoadenylate synthetase family is a group of interferon-stimulated genes that play important roles in innate antiviral defence. Recently, the minor allele of an OAS1 SNP that alters splicing was found to be associated with increased enzymatic activity and with type 1 diabetes. In this study, we investigate OAS1 SNP with SLE association in 250 nuclear families using transmission disequilibrium test.
We find:
(1) Three SNPs, rs352130, rs352140 and rsl87084 are identified in TLR9 genomic region. Two SNPs, rs4988457and rs7744 are identified in MyD88 genomic region. Furthermore, a new SNP is found in MyD88 5' regulatory region. rsl061502 and rs2277270 are identified in IRF7 coding region and regulatory region. There are also two SNPs, rs540386 and rs5030437 locate in first intron of TRAF6.
(2) The minor rs540386 T allele frequency was significantly lower in SLE cases than controls (p<0.05). There are 2.4% of SLE cases carrying the
引文
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5. Kirschning CJ, Bauer S. Toll-like receptors: celluar signal transducers for exogenous molecular patterns causing immune responses. Int J Med Microbiol. 2001, 291: 251-60.
6. Beutler B. Inferences, questions and possibilities in Toll-like receptor signaling. Nature, 2004, 430: 257-263.
7. Is targeting Toll-like receptors and their signaling pathway a useful therapeutic approach to modulating cytokine-driven inflammation? Immunol Rev, 2004, 202: 250-265.
8. Kawai T, Sato S, Ishii K J, et al. Interferon-alpha induction through Toll-like receptors involves a direct interaction of IRF7 with MyD88 and TRAF6. Nat Immunol, 2004, 5: 1061-1068.
9. Honda K, Yanai H, Mizutani T, et al. Role of a transductional-transcriptional processor complex involving MyD88 and IRF7 in Toll-like receptor signaling. PNAS, 2004, 101: 15416-15421. Seldin MF, Amos CI, Ward R, et al. The genetics revolution and assault on rheumatoid arthritis. Arthritis Rheum, 1999, 42: 1071-1079.
10. Isaacs A, Lindenmann J. Virus inference. I. The interferon. Proc R Soc Lond, 1957, 147: 258-267.
11. Ortaldo JR, Mantovani A, Hobbs D, et al. Effects of several species of human leukocyte interferon on cytotoxic activity of NK cells and monocytes, Int J Cancer, 1983, 31: 285-289.
12. Zhang X, Sun S, Hwang I, et al. Potent and selective stimulation of memory-phenotype CD8+ T cells in vivo by IL15. Immunity, 1998, 8: 591-599.
13. Marrrack P, Kappler J, Mitchell T. Type Ⅰ interferons keep activated T cells alive.??J Exp Med, 1999, 189: 521-530.
14. Le Bon A, Schiavoni G, D'Agostino G, et al. Type Ⅰ interferons petently enhance humoral immunity and can promote isotype switching by stimulating dendritic cells in vivo. Immunity, 2001, 14: 461-470.
15．汤建平，顾越英，沈南等．系统性红斑狼疮患者外周血干扰素基因的定量表达．中华内科杂志，2005，44：106-110．
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17. Bengtsson A, Sturfelt G, Truedsson L, et al. Activation of type Ⅰ interferon systemic lupus erythematosus correlates with disease activity but not antiretroviral antibodies. Lupus, 2000, 9: 664-671.
18. von Wussow P, Jakschies D, Hochkeppel H, et al. MX homologous protein in mononuclear cells from patients with systemic lupus erythematosus. Arithris Rheum, 1989, 32:914-918.
19. Bennett L, Palucka AK, Arce E, et al. Interferon and granulopoiesis signatures in systemic lupus erythematosus blood. J Exp Med, 2003, 197: 711-723.
20. Baechler EC, Batliwalla FM, Karypis G, et al. Interferon-inducible gene expression signature in peripheral blood cells of patients with severe lupus. PNAS, 2003, 100: 2610-2615.
21. Paterson AD. Genetic epidemiology of type 1 diabetes. Curr Diab Rep, 2006, 6: 139-146.
22. Balan V, Nelson DR, Sulkowski MS. A phase Ⅰ/Ⅱ study evaluating escalating doses of recombination human albumin-interferon-alpha fusion protein in chronic hepatitis C patients who have failed previous interferon-alpha-based therapy. Antivir Ther, 2006, 11: 35-45.
23. Ye S, Guo Q, Tang JP, et al. Could 2'5'-oligoadenylate synthetase isoforms be biomarkers to differentiate between disease flare and infection in lupus patients? A pilot study. Clin Rheumatol, 2006.
24. Blanco P, AK Palucka, M Gill, et al. Induction of dendritic cell differentiation by IFN-alpha in systemic lupus erythematosus. Science, 2001, 294: 1540-1543.
25. Cederblad B, Blomberg S, Vallin H, et al. Patients with systemic lupus erythematosus have reduced numbers of circulating natural interferon-alpha-producing cells. J Autoimmun, 1998, 11: 465-470.
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2. Lien E, Ingall RR. Toll-like receptors. Crit Care Med, 2002, 30: 1-11.
3. Schwarz K, Storni T, Manolova V, et al. Role of Toll-like receptors in costimulating cytotoxic T cell responses. Eur J Immunol, 2003, 33: 1465-1470.
4. Akira S, Takeda K, Kaisho T. Toll-like receptors: critical proteins linking innate and acquired immunity. Nat Immunol. 2001, 2: 675-80.
5. Kirschning CJ, Bauer S. Toll-like receptors: celluar signal transducers for exogenous molecular patterns causing immune responses. Int J Med Microbiol. 2001, 291: 251-60.
6. Beutler B. Inferences, questions and possibilities in Toll-like receptor signaling. Nature, 2004, 430: 257-263.
7. Is targeting Toll-like receptors and their signaling pathway a useful therapeutic approach to modulating cytokine-driven inflammation? Immunol Rev, 2004, 202: 250-265.
8. Kawai T, Sato S, Ishii K J, et al. Interferon-alpha induction through Toll-like receptors involves a direct interaction of IRF7 with MyD88 and TRAF6. Nat Immunol, 2004, 5: 1061-1068.
9. Honda K, Yanai H, Mizutani T, et al. Role of a transductional-transcriptional processor complex involving MyD88 and IRF7 in Toll-like receptor signaling. PNAS, 2004, 101: 15416-15421. Seldin MF, Amos CI, Ward R, et al. The genetics revolution and assault on rheumatoid arthritis. Arthritis Rheum, 1999, 42: 1071-1079.
10. Isaacs A, Lindenmann J. Virus inference. I. The interferon. Proc R Soc Lond, 1957, 147: 258-267.
11. Ortaldo JR, Mantovani A, Hobbs D, et al. Effects of several species of human leukocyte interferon on cytotoxic activity of NK cells and monocytes, Int J Cancer, 1983, 31: 285-289.
12. Zhang X, Sun S, Hwang I, et al. Potent and selective stimulation of memory-phenotype CD8+ T cells in vivo by IL15. Immunity, 1998, 8: 591-599.
13. Marrrack P, Kappler J, Mitchell T. Type Ⅰ interferons keep activated T cells alive.??J Exp Med, 1999, 189: 521-530.
14. Le Bon A, Schiavoni G, D'Agostino G, et al. Type Ⅰ interferons petently enhance humoral immunity and can promote isotype switching by stimulating dendritic cells in vivo. Immunity, 2001, 14: 461-470.
15．汤建平，顾越英，沈南等．系统性红斑狼疮患者外周血干扰素基因的定量表达．中华内科杂志，2005，44：106-110．
16. Ytterberg SR, Schnitzer TJ. Serum interferon levels in patients with systemic lupus erythematosus. Arithritis Rheum. 1982, 25: 401-406.
17. Bengtsson A, Sturfelt G, Truedsson L, et al. Activation of type Ⅰ interferon systemic lupus erythematosus correlates with disease activity but not antiretroviral antibodies. Lupus, 2000, 9: 664-671.
18. von Wussow P, Jakschies D, Hochkeppel H, et al. MX homologous protein in mononuclear cells from patients with systemic lupus erythematosus. Arithris Rheum, 1989, 32:914-918.
19. Bennett L, Palucka AK, Arce E, et al. Interferon and granulopoiesis signatures in systemic lupus erythematosus blood. J Exp Med, 2003, 197: 711-723.
20. Baechler EC, Batliwalla FM, Karypis G, et al. Interferon-inducible gene expression signature in peripheral blood cells of patients with severe lupus. PNAS, 2003, 100: 2610-2615.
21. Paterson AD. Genetic epidemiology of type 1 diabetes. Curr Diab Rep, 2006, 6: 139-146.
22. Balan V, Nelson DR, Sulkowski MS. A phase Ⅰ/Ⅱ study evaluating escalating doses of recombination human albumin-interferon-alpha fusion protein in chronic hepatitis C patients who have failed previous interferon-alpha-based therapy. Antivir Ther, 2006, 11: 35-45.
23. Ye S, Guo Q, Tang JP, et al. Could 2'5'-oligoadenylate synthetase isoforms be biomarkers to differentiate between disease flare and infection in lupus patients? A pilot study. Clin Rheumatol, 2006.
24. Blanco P, AK Palucka, M Gill, et al. Induction of dendritic cell differentiation by IFN-alpha in systemic lupus erythematosus. Science, 2001, 294: 1540-1543.
25. Cederblad B, Blomberg S, Vallin H, et al. Patients with systemic lupus erythematosus have reduced numbers of circulating natural interferon-alpha-producing cells. J Autoimmun, 1998, 11: 465-470.
26. Farkas L, Beiske K, Lund-Johansen F, et al. Plasmacytoid dendritic cells??(natural interferon-alpha/beta-producing cells) accumulate in cutaneous lupus erythematosus lesions. Am J Pathol, 2001, 159: 237-243.
27. Blomberg S, Eloranta ML, Cederblad B, et al. Presence of cutaneous interferon-alpha producing cells in patients with systemic lupus erythematosus. Lupus, 2001, 10: 484-490.
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