CN116287208A - 一种无创诊断冠状动脉扩张症的方法 - Google Patents
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Abstract
本发明属于生物医药领域,具体涉及一种无创诊断冠状动脉扩张症的方法。具体地,本发明提供了检测USP18的表达量和/或甲基化程度的试剂在制备诊断冠状动脉扩张症的产品中的应用。
Description
技术领域
本发明属于生物医药领域,具体涉及一种无创诊断冠状动脉扩张症的方法。
背景技术
冠状动脉扩张(coronary artery ectasia,CAE)是一种少见但容易识别的解剖形态学异常。一般是指心外膜下冠状动脉的弥漫性扩张,超过邻近正常节段的1.5倍,超过2倍以上的局限性扩张般被称作冠状动脉瘤。CAE可以单发,也可以多发,可为囊状(瘤体横径大于长径)或梭形(瘤体横径小于长径)。50%的CAE患者合并冠状动脉粥样硬化。单纯性CAE是指排除动脉粥样硬化、血管炎、川崎病、感染性疾病、先天性冠状动脉疾病等病因的不明原因所致者。
CAE的病因学机制尚未完全明确。病理表现主要为冠状动脉血管壁中层结构的破坏,弹力纤维降解,可能病因主要有以下几种:1、动脉粥样硬化,有学者认为CAE是阻性冠脉疾病的一种变异型。2、自身免疫或者炎症反应,儿童和青少年的CAE通常为川崎病的晚期并发症。***疾病***性动脉炎(如结节性动脉炎、大动脉炎)和马方综合征均可以导致CAE。3、血管感染性疾病如真菌性或脓毒性栓子、梅毒、疏螺旋体病等。4、单纯性CAE病因不明,可能与基因易感性(如特殊HLAT类基因型,基质金属蛋白酶基因变异)、血管紧张素转换酶过度表达等相关。
CAE诊断的金标准是冠状动脉造影。冠状动脉造影提示冠状动脉管腔的扩张程度达到CAE标准合并或者不合并冠状动脉粥样硬化斑块、狭窄、血栓。同时不伴随川崎病、***性血管炎(红斑狼疮、多发性大动脉炎、结节性多动脉炎和白塞病等)、梅毒以及冠状动脉旋磨、支架植入等介入治疗并发症等情况。冠状动脉造影大大提高了检出率,在接受冠脉造影的人群中的发病率为1.2%~9.9%,其中冠心病和腹主动脉瘤患者的检出率较高。单纯性CAE检出率为0.1%~0.32%。
高通量测序技术的快速发展和应用,为冠状动脉扩张症发病机理的研究提供了更加全面和快速的分析手段,也为冠状动脉扩张症的进一步治疗方案提供崭新思路。
发明内容
本发明通过测序获得冠状动脉扩张症患者的基因信息,结合甲基化分析结果,提供了可以诊断冠状动脉扩张病的诊断靶点USP18,所示诊断靶点USP18在患者样本中的表达量变化经荧光定量PCR验证与数据分析结果一致,又经过受试者工作特征曲线验证,证明了本发明USP18在诊断中的应用价值。
第一方面,本发明提供了检测USP18的表达量和/或甲基化程度的试剂在制备诊断冠状动脉扩张症的产品中的应用。
优选地,所述USP18在冠状动脉扩张症患者中甲基化程度低、表达量高。
优选地,所述“高”指所述USP18在患者体内的表达水平大于健康对照群体体内的表达水平,相对于对照表达水平高至少1.1倍,具体地,至少1.1倍、1.2倍、1.3倍、1.4倍、1.5倍、1.6倍、1.7倍、1.8倍、1.9倍、2.0倍、2.1倍、2.2倍、2.3倍、2.4倍、2.5倍、2.6倍、2.7倍、2.8倍、2.9倍、3.0倍、3.1倍、3.2倍、3.3倍、3.4倍或3.5倍或更多。
优选地,所述“低”指所述USP18在患者体内的甲基化程度小于健康对照群体体内的表达水平,例如是对照表达量的约10%、20%、30%、40%、50%、60%、70%、80%、90%。
优选地,所述检测是针对受试者样品进行检测。所述样品包括:组织、血液尿液、唾液、***、乳汁、脑脊髓液、泪液、痰、粘液、淋巴、胞液、腹水、胸膜积液、羊水、膀胱冲洗液和支气管肺泡灌洗液。
具体地,所述健康对照是确认不患有冠状动脉扩张症的人群。
最优选地,所述样品是血液。
优选地,所述表达量包括mRNA表达量和/或蛋白表达量。
优选地,所述检测mRNA表达量的试剂包括以下方法中使用到的试剂:基于PCR的定量检测方法、Southern杂交、Northern杂交、点杂交、荧光原位杂交(FISH)、DNA微阵列、ASO法、高通量测序平台。
具体地,所述检测mRNA表达量的试剂例如特异性引物和/或探针,如本发明具体实施例中使用荧光定量PCR对USP18进行了定量检测。
优选地,所述探针可以是DNA、RNA、DNA-RNA嵌合体、PNA或其它衍生物。所述探针的长度没有限制,只要完成特异性杂交、与目的核苷酸序列特异性结合,任何长度都可以。如本文中所使用的,术语“杂交”应当包括“核酸链经由碱基配对而与互补链结合的过程”以及如聚合酶链式反应技术中所实施的扩增过程。
当针对mRNA进行检测时,也可以使用逆转录-聚合酶链式反应(RT-PCR)进行检测;具体地,使用RNA提取技术(例如使用酸性酚/异硫氰酸胍)或成品试剂盒提取待测样品中的RNA,逆转录为DNA后再针对DNA进行表达量的检测。
优选地,前述基于PCR的检测方法也可以替换为其他扩增方法,例如:链替换扩增(Strand displacement amplification,SDA),依赖核酸序列的扩增(Nucleic acidsequence-based amplification,NASBA),转录依赖的扩增***(Transcript-basedamplification system,TAS),Q复制酶(Q-beta replicase)催化RNA扩增,滚环扩增(Rolling circle amplification,RCA),环介导的等温扩增(Loop mediated isothermalamplification,LAMP)。
优选地,所述检测蛋白表达量的试剂包括以下方法中使用到的试剂:蛋白质印迹(Western Blot法)、酶联免疫吸附测定(ELISA)、放射性免疫测定(RIA)、夹心测定、免疫组织化学染色、质谱法、免疫沉淀分析法、补体结合分析法、流式细胞荧光分边技术和蛋白质芯片。
具体地,常见的检测蛋白表达量的试剂包括特异性抗体。优选地,所述抗体包括完整的抗体分子、抗体的任何片段或带有修饰的抗体,具体地,所述抗体包括嵌合抗体、scFv、Fab、F(ab’)2、Fv等。只要所述片段能够保留与蛋白的结合能力即可。用于检测蛋白质水平的抗体的制备是本领域技术人员公知的,并且本发明可以使用任何方法来制备所述抗体。
前述任意检测方法中的检测结果可以通过标记物体现,具体地,所述标记物包括:放射性标记物、酶标记物、化学发光标记物、荧光标记物和其它合适的标记物。
优选地,所述产品包括试剂盒、诊断设备。
另一方面,本发明还提供了诊断冠状动脉扩张症的试剂盒,所述试剂盒中包括检测USP18表达量的试剂。
优选地,所述试剂盒中包括前述检测mRNA表达量的试剂。
优选地,所述试剂盒中包括前述检测蛋白表达量的试剂。
优选地,所述试剂盒中还可以包括mRNA表达量辅助检测试剂、蛋白表达量辅助检测试剂、检测仪器。
优选地,所述mRNA表达量辅助检测试剂包括但不限于:使所述引物对应的扩增子可视化的反应试剂,例如通过琼脂糖凝胶电泳法、酶联凝胶法、化学发光法、原位杂交法、荧光检测法等使扩增子可视化的试剂;RNA提取试剂;逆转录试剂;cDNA扩增试剂;制备标准曲线所用的标准品;阳性对照品;阴性对照品。
更具体地,所述阳性对照品包括取自确诊冠状动脉扩张症患者的样品,所述阴性对照品包括取自健康对照的样品,所述健康对照以确认不是冠状动脉扩张症患者。所述样品包括直接取自受试者的血液,也包括经过处理的样品,例如为易于保存进行RNA转录而得到的DNA样本等。
优选地,所述蛋白表达量辅助检测试剂包括但不限于:封闭液、抗体稀释液、洗涤缓冲液、显色终止液、制备标准曲线的标准品。
优选地,所述检测装置包括实时定量PCR仪、高通量测序平台、检测芯片和芯片信号读取器。
本发明所述合适的试剂、对照、说明书等一起在合适的容器中包装入试剂盒中。
另一方面,本发明还提供了前述试剂盒在制备诊断冠状动脉扩张症的产品中的应用。
另一方面,本发明提供了一种冠状动脉扩张症的诊断方法,所述方法包括将USP18的表达量检测结果和/或甲基化程度检测结果与阈值比较,根据比较结果判断受试者是否患有冠状动脉扩张症。
更具体地,所述USP18的表达量检测结果、甲基化程度检测结果是针对来自受试者的样本进行检测得到的。
优选地,所述样本是血液。
更具体地,所述判断的方法是比较USP18的表达量检测结果与阈值的大小,如果输入的USP18的表达量高于阈值,则代表患病;反之则未患病;如果输入的USP18的甲基化程度低于阈值,则代表患病;反之则未患病;
本发明所述术语“阈值”也可称为临界值、切值、cutoff值,在不同检测方法、检测试剂下得到的阈值是不同的,确定阈值的方法是本领域技术人员所熟知的,如本发明所使用的方法是,收集大量健康对照和患者样本,获得检测数据后绘制ROC曲线,即可获得该检测方法下的阈值。本发明所述阈值包括表达量阈值、甲基化程度阈值。
所述“阈值”的确定方法是本领域所通用的。不同的检测程序获得的阈值不同,有时同一厂商不同批号的试剂测得的阈值也会有差异,所以临床应用中需根据实验试剂、检测方法的变动随时调整阈值。
在一种具体的实施方式中,获得所述阈值的检测方法需要与针对该受试者样本的检测方法相同。
本发明所述“冠状动脉扩张症”即coronary artery ectasia(CAE),指各种原因造成冠状动脉局限性或弥漫性扩张超过其邻近正常冠脉管径的1.5倍或以上。
如本发明所述的,CAE诊断的金标准是冠状动脉造影。冠状动脉造影提示冠状动脉管腔的扩张程度达到CAE标准合并或者不合并冠状动脉粥样硬化斑块、狭窄、血栓。同时不伴随川崎病、***性血管炎(红斑狼疮、多发性大动脉炎、结节性多动脉炎和白塞病等)、梅毒以及冠状动脉旋磨、支架植入等个入治疗并发症等情况。
附图说明
图1是差异分析得到的火山图。
图2是差异分析得到的热图。
图3是差异甲基化位点的火山图。
图4是差异甲基化位点的曼哈顿图。
图5是USP18在诊断冠状动脉扩张中的受试者工作特征曲线。
具体实施方式
下面结合实施例对本发明做进一步的说明,以下所述,仅是对本发明的较佳实施例而已,并非对本发明做其他形式的限制,任何熟悉本专业的技术人员可能利用上述揭示的技术内容加以变更为同等变化的等效实施例。凡是未脱离本发明方案内容,依据本发明的技术实质对以下实施例所做的任何简单修改或等同变化,均落在本发明的保护范围内。
实施例1:数据分析及验证
步骤1、样本收集及测序数据处理
对收集到的血液样本进行测序,本发明涉及的样本信息如表1所示。基于边合成边测序(Sequencing By Synthesis,SBS)技术,使用Illumina HiSeq高通量测序平台对cDNA文库进行测序,能够产出大量的高质量Reads,测序平台产出的这些Reads或碱基称为原始数据(Raw Data)。通过Illumina平台,得到了大量的样本双端测序数据。鉴于数据错误率对结果的影响,采用Trimmomatic软件对原始数据进行质量预处理,并对整个质控过程中的reads数进行统计汇总,汇总结果如表2所示。
表1、正常对照及CAE患者的样本信息
表2、测序数据统计表
注:(1)Sample:样本名称;(2)Rawreads:原始reads数目;(3)Rawbases:原始测序量,即碱基数目;(4)CleanReads:过滤后得到的cleanreads数目;(5)CleanBases:过滤后得到的测序量,碱基数目;(6)ValidBases:有效碱基百分比(7)Q30:计算Phred数值大于30的碱基占总体碱基的百分比;(8)GC:计算碱基G和C的数量总和占总的碱基数量的百分比
转录组测序数据中,只有比对到参考基因组上的数据才能用于后续分析。因此,将比对到指定的参考基因组上的Reads称为Mapped Reads。
表3、各样本mapping比对效率统计
注:sample:样本名称;total_reads:测序数据在质控后的cleanreads数;total_map:比对到基因组上的reads数及其百分比;read1_map:比对到参考基因组的read1数及其百分比;read2_map:比对到参考基因组的read2数及其百分比;splice_map:拆分比对到基因组上的reads数及其百分比;unsplice_map:未拆分比对到基因组上的reads数及其百分比proper_map:成对的read1和read2同时比对到基因组的reads数及其百分比
用已知的参考基因序列以及注释文件作为数据库,采取序列相似性比对的方法鉴定出各蛋白编码基因在各样本中的表达丰度。使用htseq-count软件获取每个样本中比对到蛋白编码基因上的reads数。比对获得counts后,需要对蛋白编码基因进行过滤,去除reads数为零的基因。各样本中检出基因数目如表4。
表4、检出基因数目统计部分结果展示
对两组mRNA的测序结果进行差异分析,首先根据counts均值对基因进行过滤,只保留counts均值大于2的基因进行下一步分析。利用DESeq2对各个样本基因的counts数目进行标准化处理(采用BaseMean值来估算表达量),计算差异倍数,并采用NB(负二项分布检验的方式)进行差异显著性检验,最终根据差异倍数及差异显著性检验结果来筛选差异蛋白编码基因。筛选差异的条件为p<0.05&|log2foldChange|>1。经分析得到152个差异表达基因,包括93个上调,59个下调。差异分析得到的火山图及热图如图1、2所示。
步骤2、差异甲基化分析
从GEO数据库下载GSE87016数据集,该数据集包含23个样本的甲基化数据(NOR:CAE=12:11),采用CHAMP包对甲基化数据进行差异甲基化分析。设置的筛选标准是P.Value<0.05,得到9377个差异甲基化位点,共4318个差异甲基化基因,包括2289个高甲基化基因,2029个低甲基化基因。差异甲基化位点的火山图和曼哈顿图如图3、4所示。
步骤3、筛选异常甲基化修饰的差异表达基因
对mRNA差异表达基因和差异甲基化基因取交集以获得异常甲基化调控的差异表达基因,得到9个高甲基化修饰表达下调的基因和11个低甲基化修饰的表达上调基因。
步骤4、荧光定量PCR验证
基于前述分析结果,USP18在CAE患者中低甲基化且高表达。
以USP18作为候选基因(病例组vs对照组),收集冠状动脉扩张症患者血液和对照血液(>15例),提取RNA样本,利用荧光定量PCR(qRT-PCR)验证候选基因在疾病组和对照组的差异表达。
步骤5、ROC曲线验证
受试者工作特征曲线(receiver operating characteristic curve)简称ROC曲线,又称感受性曲线(sensitivity curve);ROC曲线上各点反映的都是相同的感受性,通过对疾病组和参照组的测定结果进行分析,确定测定值的上下限、组距以及截断点,按选择的组距间隔列出累积频数分布表,并分别计算出所有截断点的真阳性率(敏感度)、特异性和假阳性率(1-特异性),作图绘成ROC曲线。灵敏度(sensitivity),即敏感度,是指筛检方法能将实际有病的人正确地判定为患者的比例。特异度(specificity),是指筛检方法能将实际无病的人正确地判定为非患者的比例。
根据USP18获得的ROC曲线中,AUC值为0.76,敏感度为0.769,1-特异度为0.25,具体如图5所示。
以上所述实施例的各技术特征可以进行任意的组合,为使描述简洁,未对上述实施例中的各个技术特征所有可能的组合都进行描述,然而,只要这些技术特征的组合不存在矛盾,都应当认为是本说明书记载的范围。
Claims (10)
1.检测USP18的表达量和/或甲基化程度的试剂在制备诊断冠状动脉扩张症的产品中的应用。
2.如权利要求1所述的应用,所述USP18在冠状动脉扩张症患者中甲基化程度低、表达量高。
3.如权利要求1所述的应用,所述检测是针对受试者样品进行检测,所述样品是血液。
4.如权利要求1所述的应用,所述表达量包括mRNA表达量和/或蛋白表达量。
5.如权利要求4所述的应用,所述检测mRNA表达量的试剂包括以下方法中使用到的试剂:基于PCR的定量检测方法、Southern杂交、Northern杂交、点杂交、荧光原位杂交、DNA微阵列、ASO法、高通量测序平台。
6.如权利要求5所述的应用,所述检测mRNA表达量的试剂例如特异性引物和/或探针。
7.如权利要求4所述的应用,所述检测蛋白表达量的试剂包括以下方法中使用到的试剂:蛋白质印迹、酶联免疫吸附测定、放射性免疫测定、夹心测定、免疫组织化学染色、质谱法、免疫沉淀分析法、补体结合分析法、流式细胞荧光分边技术和蛋白质芯片。
8.如权利要求7所述的应用,所述检测蛋白表达量的试剂包括特异性抗体。
9.诊断冠状动脉扩张症的试剂盒,所述试剂盒中包括检测USP18表达量的试剂;
优选地,所述试剂盒中包括权利要求5所述的检测mRNA表达量的试剂;
优选地,所述试剂盒中包括权利要求7所述的检测蛋白表达量的试剂;
优选地,所述试剂盒中还可以包括mRNA表达量辅助检测试剂、蛋白表达量辅助检测试剂、检测仪器;
优选地,所述mRNA表达量辅助检测试剂包括:使所述引物对应的扩增子可视化的反应试剂、RNA提取试剂、逆转录试剂、cDNA扩增试剂、制备标准曲线所用的标准品、阳性对照品、阴性对照品;
优选地,所述蛋白表达量辅助检测试剂包括:封闭液、抗体稀释液、洗涤缓冲液、显色终止液、制备标准曲线的标准品;
优选地,所述检测装置包括实时定量PCR仪、高通量测序平台、检测芯片和芯片信号读取器。
10.权利要求9所述的试剂盒在制备诊断冠状动脉扩张症的产品中的应用。
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Address after: 050200 No.3 Xingyuan Road, Luquan District, Shijiazhuang City, Hebei Province Applicant after: Hebei University of Traditional Chinese Medicine Address before: 050200 No.3 Xingyuan Road, Luquan District, Shijiazhuang City, Hebei Province Applicant before: Hebei College of Traditional Chinese Medicine |