Fat-1基因多态性与脊柱结核易感关联性研究
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摘要
目的寻找和分析与脊柱结核易感性相关的新的候选基因。方法收集2013年6月—2015年12月杭州市红十字会医院收治的脊柱结核患者193例为病例组,同时选取同时期本院体检健康者207例为对照组,采用全基因组外显子技术捕获6例脊柱结核患者外显子区DNA后,进行高通量测序,并结合现代生物信息学方法进行分析,结果初步筛选到Fat-1基因多态性(rs1280098,c.8798A>C,p.Q2933P)与脊柱结核的发生相关;随后,采用Sanger测序法对对照组和病例组患者做上述突变位点的验证。检测Fat-1基因型分布,采用实时荧光反转录聚合酶链式反应(RT-q PCR)法检测Fat-1基因相对表达水平,酶联免疫吸附试验(ELISA)法检测血清白介素(IL)-12和γ-干扰素(IFN-γ)含量,同时检测生化指标。结果病例组AA基因型129例(66.8%)、AC基因型33例(17.1%)、CC基因型31例(16.1%);对照组分别为157例(75.8%)、35例(16.9%)、15例(7.3%)。两组Fat-1基因型分布比较,差异有统计学意义(χ~2=7.885,P=0.019)。不同遗传模型的Logistic回归分析结果显示:在相加模型中,CC纯合突变基因型与脊柱结核是否发病相关,AC杂合突变基因型与脊柱结核是否发病不相关(P>0.05);在显性模型中,AC+CC突变基因型与脊柱结核是否发病相关(P<0.05);在隐性模型中,CC纯合突变基因型与脊柱结核是否发病相关(P<0.05);且该多态性位点最小等位基因C与脊柱结核是否发病相关(P<0.05)。病例组患者外周血Fat-1基因相对表达水平低于对照组,血清IL-12和IFN-γ含量高于对照组,脂蛋白A、C反应蛋白、红细胞沉降率和嗜酸粒细胞计数高于对照组,清蛋白水平低于对照组(P<0.05)。结论 Fat-1基因rs1280098位点多态性与脊柱结核的发病相关。此外,Fat-1基因该位点的突变导致Fat-1基因转录水平下调,以及血清炎性因子IL-12和IFN-γ水平上调,并最终导致脊柱结核易感性增加。这些检测结果为脊柱结核的早期诊断提供了分子依据。
Objective To find and explore new candidate genes associated with spinal tuberculosis susceptibility.Methods A total of 193 patients with spinal tuberculosis,who were treated in Hangzhou Red Cross Hospital from June 2013 to December 2015,were selected as the case group.A further 207 healthy subjects who came for physical tests in the same hospital during the same period were selected as the control group.We used high-throughput sequencing to analyze DNA from six patients and used bioinformatics to determine candidate genes associated with tuberculosis. Results showed that the gene polymorphism,Fat-1(rs1280098,c.8798 A > C,p.Q2933 P),was associated with the occurrence of spinal tuberculosis.Subsequently,the Sanger sequencing method was used to verify this mutation loci in both the control group and case group.Expression of Fat-1 was subsequently detected using real-time fluorescent reverse transcription polymerase chain reaction(qRT-PCR). In addition,the levels of interleukin(IL)-12 and interferon gamma(IFN-γ),as well as other biochemical indicators were detected. Results There were 129(66.8%) AA genotypes,33(17.1%) AC genotypes,and 31(16.1%) CC genotypes in the case group,while there were 157(75.8%) AA genotypes,35(16.9%) AC genotypes,and 15(7.3%) CC genotypes in the control group.There was a significant difference between the two groups in the distribution of Fat-1 genotype(χ~2=7.885,P=0.019). Logistic regression analysis of different genetic models showed that,in the additive model,CC homozygous mutation genotypes were correlated with increased incidences of spinal tuberculosis,while AC heterozygous mutation genotypes were associated with normal incidences of spinal tuberculosis(P>0.05). In the dominant model,AC+CC mutation genotypes were correlated with increased incidences of spinal tuberculosis(P<0.05);in the recessive model,CC homozygous mutation genotypes were correlated with increased incidences of spinal tuberculosis(P<0.05);and the minor allele C of the polymorphism locus was related to the incidence of spinal tuberculosis(P<0.05). The expression of Fat-1 in peripheral blood was lower in the case group than in the control group,while the levels of serum IL-12 and IFN-γ were higher in the case group compared with the control group(P<0.05). The levels of lipoprotein A,C-reactive protein,erythrocyte sedimentation rate,and eosinophil counts in the case group were higher than those in the control group,while albumin level in the case group was lower than that in the control group(P<0.05). Conclusion The gene polymorphism,Fat-1,is associated with the occurrence of spinal tuberculosis.In addition,the mutation of the Fat-1 gene results in reduced levels of Fat-1 gene transcription,as well as increased levels of the inflammatory cytokines,IL-12 and IFN-γ,in serum.These test results provide a molecular mechanism for the early diagnosis of spinal tuberculosis.
Objective To find and explore new candidate genes associated with spinal tuberculosis susceptibility.Methods A total of 193 patients with spinal tuberculosis,who were treated in Hangzhou Red Cross Hospital from June 2013 to December 2015,were selected as the case group.A further 207 healthy subjects who came for physical tests in the same hospital during the same period were selected as the control group.We used high-throughput sequencing to analyze DNA from six patients and used bioinformatics to determine candidate genes associated with tuberculosis. Results showed that the gene polymorphism,Fat-1(rs1280098,c.8798 A > C,p.Q2933 P),was associated with the occurrence of spinal tuberculosis.Subsequently,the Sanger sequencing method was used to verify this mutation loci in both the control group and case group.Expression of Fat-1 was subsequently detected using real-time fluorescent reverse transcription polymerase chain reaction(qRT-PCR). In addition,the levels of interleukin(IL)-12 and interferon gamma(IFN-γ),as well as other biochemical indicators were detected. Results There were 129(66.8%) AA genotypes,33(17.1%) AC genotypes,and 31(16.1%) CC genotypes in the case group,while there were 157(75.8%) AA genotypes,35(16.9%) AC genotypes,and 15(7.3%) CC genotypes in the control group.There was a significant difference between the two groups in the distribution of Fat-1 genotype(χ~2=7.885,P=0.019). Logistic regression analysis of different genetic models showed that,in the additive model,CC homozygous mutation genotypes were correlated with increased incidences of spinal tuberculosis,while AC heterozygous mutation genotypes were associated with normal incidences of spinal tuberculosis(P>0.05). In the dominant model,AC+CC mutation genotypes were correlated with increased incidences of spinal tuberculosis(P<0.05);in the recessive model,CC homozygous mutation genotypes were correlated with increased incidences of spinal tuberculosis(P<0.05);and the minor allele C of the polymorphism locus was related to the incidence of spinal tuberculosis(P<0.05). The expression of Fat-1 in peripheral blood was lower in the case group than in the control group,while the levels of serum IL-12 and IFN-γ were higher in the case group compared with the control group(P<0.05). The levels of lipoprotein A,C-reactive protein,erythrocyte sedimentation rate,and eosinophil counts in the case group were higher than those in the control group,while albumin level in the case group was lower than that in the control group(P<0.05). Conclusion The gene polymorphism,Fat-1,is associated with the occurrence of spinal tuberculosis.In addition,the mutation of the Fat-1 gene results in reduced levels of Fat-1 gene transcription,as well as increased levels of the inflammatory cytokines,IL-12 and IFN-γ,in serum.These test results provide a molecular mechanism for the early diagnosis of spinal tuberculosis.
引文
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[14]刘忠泉,宋长兴,胡秀龄,等.HLA-DR.DQ基因与骨关节结核的易感性研究[J].中国防痨杂志,2000,22(3):182-187.DOI:10.3969/j.issn.1000-6621.2000.03.007.LIU Z Q,SONG C X,HU X L,et al.Study of HLA-DR.DQgenes associated with tuberculosis of the bone and joint[J].Chinese Journal of Antituberculosis,2000,22(3):182-187.DOI:10.3969/j.issn.1000-6621.2000.03.007.
[15]MOON M S.Tuberculosis of the spine.Controversies and a new challenge[J].Spine,1997,22(15):1791-1797.
[2]GULLAND A.More cases of tuberculosis than previously thought,WHOreports[J].BMJ,2016,355:i5562.DOI:10.1136/bmj.i5562.
[3]DYE C,WILLIAMS B G.The population dynamics and control of tuberculosis[J].Science,2010,328(5980):856-861.DOI:10.1126/science.1185449.
[4]LI X W,YANG Y,ZHOU F,et al.SLC11A1(NRAMP1)polymorphisms and tuberculosis susceptibility:updated systematic review and meta-analysis[J].PLo S One,2012,6(1):e15831.DOI:10.1371/journal.pone.0015831.
[5]高琪乐,张宏其,郭超峰,等.MCP-1-2518位点基因多态性对脊柱结核易感性的影响[J].中国脊柱脊髓杂志,2013,23(7):654-658.DOI:10.3969/j.issn.1004-406X.2013.07.15.GAO Q L,ZHANG H Q,GUO C F,et al.Influence of monocyte chemoattactant protein-1-2518 gene polymorphism on susceptibility of spinal tuberculosi[J].Chinese Journal of Spine and Spinal Cord,2013,23(7):654-658.DOI:10.3969/j.issn.1004-406X.2013.07.15.
[6]ROTH D E,SOLO G,ARENAS F,et al.Association between vitamin D receptor gene polymorphisms and response to treatment of pulmonary tuberculosis[J].J Infect Dis,2004,190(5):920-927.DOI:10.1086/423212.
[7]TSO H W,LAU Y L,TAM C M,et al.Associations between IL12B poly-morphisms and tuberculosis in the Hong Kong Chinese population[J].J Infect Dis,2004,190(5):913-919.DOI:10.1086/422693.
[8]World Health Organization.Global tuberculosis report2016[R/OL].(2016-10-14)[2017-02-15].http://apps.who.int/iris/bitstream/10665/250441/1/9789241565394-eng.pdf?ua=1&ua=1.
[9]LISTED N.The tumor suppressor FAT1 modulates WNT activity in multiple cancers[J].Cancer Discov,2013,3(3):OF19.DOI:10.1158/2159-8290.CD-RW2013-031.
[10]MORRIS L G,KAUFMAN A M,GONG Y,et al.Recurrent somatic mutation of FAT1 in multiple human cancers leads to aberrant Wnt activation[J].Nat Genet,2013,45(3):253-261.DOI:10.1038/ng.2538.
[11]MAEDA K,KOBAYASHI Y,UDAGAWA N,et al.Wnt5a-Ror2signaling between osteoblast-lineage cells and osteoclast precursors enhances osteoclastogenesis[J].Nat Med,2012,18(3):405-412.DOI:10.1038/nm.2653.
[12]MIAO C G,YANG Y Y,HE X,et al.Wnt signaling pathway in rheumatoid arthritis,with special emphasis on the different roles in synovial inflammation and bone remodeling[J].Cell Signal,2013,25(10):2069-2078.DOI:10.1016/j.cellsig.2013.04.002.
[13]BELLAMY R.Interferon-gamma and host susceptibility to tuberculosis[J].Am J Respir Crit Care Med,2003,7:946-947.DOI:10.1164/rccm.2301005.
[14]刘忠泉,宋长兴,胡秀龄,等.HLA-DR.DQ基因与骨关节结核的易感性研究[J].中国防痨杂志,2000,22(3):182-187.DOI:10.3969/j.issn.1000-6621.2000.03.007.LIU Z Q,SONG C X,HU X L,et al.Study of HLA-DR.DQgenes associated with tuberculosis of the bone and joint[J].Chinese Journal of Antituberculosis,2000,22(3):182-187.DOI:10.3969/j.issn.1000-6621.2000.03.007.
[15]MOON M S.Tuberculosis of the spine.Controversies and a new challenge[J].Spine,1997,22(15):1791-1797.