CN107904302A - 一组同时检测抗凝药物相关基因多态性的引物及应用 - Google Patents
一组同时检测抗凝药物相关基因多态性的引物及应用 Download PDFInfo
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- CN107904302A CN107904302A CN201711221471.5A CN201711221471A CN107904302A CN 107904302 A CN107904302 A CN 107904302A CN 201711221471 A CN201711221471 A CN 201711221471A CN 107904302 A CN107904302 A CN 107904302A
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Abstract
本发明公开了一组同时检测抗凝药物相关基因多态性的引物,其包括PCR扩增引物和SNaPshot PCR引物;同时检测的位点包括VKORC1‑1639 G>A、CYP4F2*3、CYP2C9*3、 CYP2C19*2、CYP2C19*3、CYP2D6*10;采用上述引物进行检测,与常规的对多个位点进行单独扩增相比,不仅减少了扩增程序,降低了扩增成本,同时保证了高灵敏度、准确度以及精密度,保证了该方法的可靠性,有助于医生为患者正确选择药物并合理调整药物剂量。
Description
技术领域
本发明属于基因多态性检测领域,具体涉及一种同时检测VKORC1-1639 G>A、 CYP4F2*3、CYP2C9*3、CYP2C19*2、CYP2C19*3、CYP2D6*10基因多态性的引物与应用。
背景技术
导致心血管疾病高发的原因之一就是血栓的形成,抗凝药及抗血小板药就是两类广泛用于抗栓的药物,在心血管疾病的治疗中占主导地位,心脑血管血栓治疗和心脑血管疾病介入术治疗过程中都需要用到这两类抗栓药,临床最常用的两类药物为华法林及氯吡格雷。基于其抗栓的作用机制,如果使用不当,可能导致出血风险,并危及患者生命。华法林和氯吡格雷均需在机体内经细胞色素CP450药物代谢相关酶类代谢后才可产生其活性成分并发挥抗栓作用;其中华法林需经CYP4F2、CYP2C9代谢,而VKORC1的多态性亦会影响华法林的作用效果;而氯吡格雷主要经CYP2C19代谢产生抗血小板的活性成分。FDA及抗栓治疗的相关指南建议,在使用华法林的过程中,需对CYP4F2、CYP2C9和VKORC1基因型进行检测,并代入华法林剂量计算公式计算初始用药剂量;而氯吡格雷使用过程中,最好对CYP2C19的基因型进行检测,对慢代谢型基因型(CYP2C19*2、CYP2C19*3)患者,需增加氯吡格雷的剂量,或选用其他不经CYP2C19代谢的抗血小板药物如替格瑞洛等。
维生素K氧化还原酶是抗凝药物华法林的作用靶点。维生素K环氧化物还原酶复合物1的编码基因VKORC1的遗传变异可通过影响VKORC1表达,从而影响华法林的敏感性。位于该基因启动子区(-1639 G>A)的单核苷酸突变 rs9923231可影响VKORC1的表达,是导致华法林用药剂量个体差异的主要原因之一。与该位点AA基因型患者相比,-1639GA和GG基因型患者平均华法林剂量分别增加52%(95% CI:41~64%)和102%(95% CI:85~118%)。VKORC1多态性对华法林剂量影响的比重因种族而异,-1639GA和GG基因型对白种人华法林剂量的影响比对亚洲人的影响分别高10%和50%。总体上,VKORC1多态性在不同种族不同人群中可解释约27%华法林用药剂量的个体差异。VKORC1 -1639A等位基因在亚洲人、白种人和黑种人群中的等位基因频率分别为91.17%、38.79%和10.81%(根据千人数据库的结果:在亚洲人、白种人和黑种人群中的等位基因频率分别为92%、40%和7%),其频率分布的种族差异与华法林用药剂量差异间具有很好的相关性。VKORC多态性同时也影响华法林用药的临床后果。美国FDA于2007年批准修改华法林的产品说明书,推荐在使用华法林前对VKORC1进行基因检测;2010年再次修改说明书,建议结合VKORC1和CYP2C9基因型考虑华法林的初始用药剂量。临床上也可根据考虑了VKORC1和CYP2C9基因型、年龄、身高、体重、种族、是否合用肝药酶诱导剂和是否合用胺碘酮等因素的剂量计算公式确定华法林初始用药剂量。
CYP4F2为维生素K单氧酶,可氧化底物生成ω-羟基衍生物。CYP4F2*3(rs2108622C>T,V433M)可导致酶活性降低,野生型纯合子基因型个体代谢活性最高,CYP4F2*3杂合子其次,CYP4F2*3纯合子活性最低。CYP4F2*3纯合子个体酶活性下降导致维生素K浓度升高,华法林的抗凝效果增强。临床研究提示,CYP4F2*3多态性与华法林稳态剂量相关,可解释1~10%的华法林剂量个体差异。携带CYP4F2*3等位基因的个体应用华法林时出血的风险显著增加。CPIC指南建议降低CYP4F2*3纯合子基因型个体华法林及香豆素类抗凝药(醋硝香豆素、苯丙香豆素)的用药剂量。
CYP2C19参与氯吡格雷、S-美芬妥英、奥美拉唑、伏立康唑、安定、去甲安定等药物的代谢。CYP2C19遗传变异可导致酶活性的个体差异,使人群出现超快代谢者(ultrarapidmetabolizer,UM)、快代谢者(extensive metabolizer,EM)、中间代谢者(intermediatemetabolizer,IM)和慢代谢者(poor metabolizer,PM)4种表型。CYP2C19*2(rs4244285,c.681G>A)和CYP2C19*3(rs4986893,c.636G>A)是中国人群中存在的2种导致CYP2C19酶缺陷的主要等位基因。CYP2C19*2导致剪接缺失,CYP2C19*3为终止密码子突变。EM个体只携带CYP2C19*1等位基因,IM个体携带CYP2C19*2或CYP2C19*3杂合子基因型;PM个体包括CYP2C19*2/*2、CYP2C19*2/*3和CYP2C19*3/*3基因型。东方人群中75~85%的PM由CYP2C19* 2所致,约20~25%的PM由CYP2C19*3所致。
氯吡格雷是一种抗血小板药物,广泛用于急性冠脉综合征、缺血性脑血栓、闭塞性脉管炎和动脉硬化及血栓栓塞引起的并发症。心脏支架手术后的患者需长期服用氯吡格雷以防止支架内再梗。氯吡格雷主要经CYP2C19代谢活化后发挥抗血小板效应。CYP2C19 PM患者应用常规剂量的氯吡格雷后体内活性代谢物生产减少,对血小板的抑制作用下降。美国FDA和美国心脏病学会建议,对于CYP2C19慢代谢基因型患者需考虑改变治疗方案,具体意见为:CYP2C19*1/*1基因型个体应用氯吡格雷有效,可常规使用;CYP2C19*2或*3基因型个体对氯吡格雷疗效降低,建议更换成普拉格雷或替卡格雷;CYP2C19*2或*3突变型纯合子个体应用氯吡格雷效果差,建议换用普拉格雷或替卡格雷。
阿米替林为三环类抗抑郁药,主要用于焦虑性或激动性抑郁症的治疗。阿米替林在体内主要经CYP2C19代谢为活性代谢产物去甲替林。CYP2C19活性的高低可通过影响血液中阿米替林与去甲替林的浓度比,影响阿米替林的疗效和不良反应的产生。CYP2C19 PM个体血浆阿米替林与去甲替林浓度的比值显著升高,5-羟色胺再摄取的抑制作用显著增强。由于三环类抗抑郁药具有多种不良反应如抗胆碱作用、中枢神经系统不良反应和心血管不良反应,与治疗失败密切相关。调整携带CYP2C19突变等位基因患者阿米替林的起始用药剂量有助于降低初始治疗的失败率。CPIC指南建议CYP2C19 EM和IM基因型患者应用常规起始剂量的阿米替林,而CYP2C19 PM基因型个体阿米替林的起始剂量应降低至常规剂量的50%,并进行治疗药物监测。
伏立康唑是一种广谱三唑类抗真菌药,CYP2C19是其主要代谢酶之一。CYP2C19 EM与PM个体间伏立康唑的血液浓度存在显著差异,PM个体在应用常规剂量药物时可能出现毒副反应,建议减少用药剂量;EM和IM个体可给予常规剂量。在常规剂量治疗时,若EM个体出现毒副反应或PM疗效不佳,均应考虑更换药物。FDA批准的药物说明书中指出应用伏立康唑前需检测CYP2C19基因型,以确保用药安全。
CYP2D6又称异喹胍4’-羟化酶, CYP第二亚家族中的重要成员。人群中CYP2D6的活性呈现强代谢者(EM)、中间代谢者(IM)、弱代谢者(PM)和超强代谢者(UM)四态分布的现象。白种人群中CYP2D6 PM的发生率高达5~10%,而在东方人群中PM的发生率约为1%。
目前已发现了CYP2D6基因的70多种遗传变异。不同突变类型对酶活性和药物代谢的影响不一。中国人群中CYP2D6常见的导致酶活性降低的等位基因包括CYP2D6*3(A2637deletion)、CYP2D6*4(G1934A)、CYP2D6*5(CYP2D6 deletion)和CYP2D6*10(C188T),等位基因频率分别为1%、1%、6%和53%。CYP2D6*10为该酶第34位脯氨酸被丝氨酸所替代所致,导致IM表型。
导致CYP2D6酶活性缺失的多态性可影响安替比林、可待因、β受体阻滞剂如美托洛尔和卡维地洛、氯丙咪嗪、去甲替林、地昔帕明、多虑平、丙咪嗪、马普替林、奥匹哌醇、三甲丙咪嗪、昂丹司琼、曲马多和他莫昔芬等的体内代谢,从而影响这些药物的疗效和不良反应的发生,临床需根据个体的基因型进行剂量的调整。
他莫昔芬通过与雌激素竞争结合雌激素受体,从而抑制乳腺癌细胞的增殖,广泛应用于雌激素受体阳性乳腺癌的治疗。他莫昔芬主要通过其活性代谢产物4-羟他莫昔芬和吲哚昔芬发挥作用,其活性产物抑制细胞增殖的活性是他莫昔芬的100倍以上;CYP2D6活性下降可导致他莫昔芬的疗效下降;美国FDA建议雌激素受体阳性的乳腺癌患者在接受他莫昔芬治疗前进行CYP2D6基因型检测,以确保药物的疗效。
CYP2D6可将三环类抗抑郁药阿米替林代谢为无活性的代谢产物,因此IM和PM个体血浆中阿米替林的浓度升高;同时,CYP2D6也是阿米替林活性代谢物去甲替林的主要代谢酶。CPIC指南建议EM基因型个体使用常规剂量的阿米替林,IM基因型个体阿米替林的起始剂量降低至常规剂量的75%,PM基因型个体选用其他不经CYP2D6代谢的药物,或将阿米替林的起始剂量降低至常规起始剂量的50%,以避免不良反应的发生。
为此,本发明将针对影响临床心血管疾病治疗中最常用的抗凝药华法林及抗血小板药物氯吡格雷的疗效的5个关键基因:CYP4F2、CYP2C9、VKORC1、CYP2C19、CYP2D6,6个多态性位点:CYP4F2*3、CYP2C9*3、VKORC1-1639 G>A、CYP2C19*2/*3、CYP2D6*10,利用SNaPshot法同时对该6个位点进行检测。
发明内容
本发明的目的是为了克服以上现有技术的不足而提供一组同时检测VKORC1-1639 G>A、CYP4F2*3、CYP2C9*3、CYP2C19*2、CYP2C19*3、CYP2D6*10基因多态性引物;
针对VKORC1-1639 G>A PCR扩增的正向引物如SEQ ID NO:1所示,反向引物如SEQ IDNO:2所示,SNaPshot PCR引物如SEQ ID NO:13所示;
针对CYP4F2*3 PCR扩增的正向引物如SEQ ID NO:3所示,反向引物如SEQ ID NO:4所示,SNaPshot PCR引物如SEQ ID NO:14所示;
针对CYP2C9*3 PCR扩增的正向引物如SEQ ID NO:5所示,反向引物如SEQ ID NO:6所示,SNaPshot PCR引物如SEQ ID NO:15所示;
针对CYP2C19*2 PCR扩增的正向引物如SEQ ID NO:7所示,反向引物如SEQ ID NO:8所示,SNaPshot PCR引物如SEQ ID NO:16所示;
针对CYP2C19*3 PCR扩增的正向引物如SEQ ID NO:9所示,反向引物如SEQ ID NO:10所示,SNaPshot PCR引物如SEQ ID NO:17所示;
针对CYP2D6*10 PCR扩增的正向引物如SEQ ID NO:11所示,反向引物如SEQ ID NO:12所示,SNaPshot PCR引物如SEQ ID NO:18所示。
本发明同时检测VKORC1-1639 G>A、CYP4F2*3、CYP2C9*3、CYP2C19*2、CYP2C19*3、 CYP2D6*10基因多态性的SNaPshot检测方法,首先提取人体外周血白细胞基因组DNA,将其浓度定量到50ng/μL,然后通过多重PCR法扩增VKORC1-1639 G>A、CYP4F2*3、CYP2C9*3、 CYP2C19*2、CYP2C19*3、CYP2D6*10所在序列,扩增产物纯化后将对应序列进行SNaPshotPCR扩增,扩增产物纯化后通过毛细管电泳法检测荧光信号并通过软件分析确定SNP位点及基因型。
上述检测方法具体包括如下步骤:
(1)DNA提取:采用酚氯仿法提取临床上需要长期或者短期使用抗凝血药物患者的外周血样本中白细胞基因组DNA,通过检测所提DNA浓度,并将其稀释到50ng/μL;
(2)多重PCR法扩增VKORC1-1639 G>A、CYP4F2*3、CYP2C9*3、CYP2C19*2、CYP2C19*3、 CYP2D6*10所在序列,25μL反应体系中包含:ddH2O 9.5μL、2xMultiplex Buffer 12.5μL、Multiplex DNA Polymerase 1μL、Primer Mix 1μL,最后在体系中分别加入1μL 50ng/μL的DNA模板;混匀后将PCR反应管置于PCR仪上并将反应程序设置如下:1:95℃,5min;2:95℃,30s;58℃,90s;72℃,90s;该步骤循环35次;3:72℃,10min;4:4℃;
(3)多重PCR产物纯化:取15μL步骤(2)中的产物,加入5UCIAP及2UExol,混合均匀后置于PCR仪,按如下程序反应:1:37℃,1h;2:75℃,15min;3:4℃;纯化的产物稀释到200ng/μL;
(4)SNaPshot PCR扩增:SNaPshot PCR反应体系为:ddH2O 3.5μL、5xSequencingBuffer 2μL、Multiplex Ready Reaction Mix 0.5μL、PrimerMix 1μL、步骤(3)中纯化后稀释的产物3μL;其中引物混合物的比例如下:SBE-VKORC1-1639 G>A:SBE-CYP4F2*3:SBE-CYP2C9*3:SBE-CYP2C19*2:SBE-CYP2C19*3:SBE-CYP2D6*10=4:4:12:1:2:2;将上述体系充分混匀微离,置入PCR仪执行如下程序:1:96℃,10s;55℃,5s;60℃,30s;循环25次;2:4℃;
(5)SNaPshotPCR扩增产物纯化:向SNaPshotPCR扩增产物中加入1UCIAP酶,按以下程序进行反应:1:37℃,60min;2:75℃,15min;3:4℃;
(6)SNaPshot分析:将步骤(5)获得的产物用ABI3500-Dx基因分析仪毛细管电泳法检测荧光信号,通过GENEMAPPER5软件分析确定基因型。
本发明另一目的是将上述引物应用在抗凝血相关药物筛选中。
本发明提供的同时检测VKORC1-1639 G>A、CYP4F2*3、CYP2C9*3、CYP2C19*2、 CYP2C19*3、CYP2D6*10基因多态性SNaPshot检测方法通过采用多重PCR对6个SNP位点所在的基因片段进行扩增程序,同时采用SNaPshot技术进行检测,与常规的对多个位点进行单独扩增和检测的方法相比,不仅减少了扩增程序,降低了扩增成本,同时保证了高灵敏度,准确度及精密度,保证了该检测方法的可靠性。
本发明提供的检测方法能够在临床上抗凝治疗相关药物的使用前进行VKORC1- 1639 G>A、CYP4F2*3、CYP2C9*3、CYP2C19*2、CYP2C19*3、CYP2D6*10基因多态性检测,判定患者对药物代谢及反应性的类型,有助于医生为患者正确选择药物并合理调整药物剂量。
附图说明
图1为VKORC1-1639 G>A、CYP4F2*3、CYP2C9*3、CYP2C19*2、CYP2C19*3、CYP2D6*10基因扩增产物测序图;
图2为SNaPshot法检测VKORC1-1639 G>A、CYP4F2*3、CYP2C9*3、CYP2C19*2、CYP2C19*3、 CYP2D6*10的图。
具体实施方式
实施例1:VKORC1-1639 G>A、CYP4F2*3、CYP2C9*3、CYP2C19*2、CYP2C19*3、CYP2D6* 10基因多态性SNaPshot检测
本检测方法是通过多重PCR法扩增待检测SNP位点所在基因片段后,采用荧光标记单碱基延伸技术(SNaPshot)结合毛细管电泳技术进行基因多态性检测,检测结果应用GENEMAPPER软件进行分析,根据峰的移动位置确定延伸产物对应的SNP位点,根据峰的荧光信号确定该SNP位点的基因型。
本检测方法可用于检测来源于EDTA抗凝外周血的DNA样本,采用多重PCR法扩增待检测SNP位点所在基因片段后,采用荧光标记单碱基延伸技术(SNaPshot)结合毛细管电泳技术进行基因多态性检测,检测结果应用GENEMAPPER软件进行分析,根据峰的移动位置确定延伸产物对应的SNP位点,根据峰的荧光信号确定该SNP位点的基因型。
本检测方法使用的主要试剂及仪器如下:
(1)引物
本检测方法所用引物均由自行设计;PCR引物序列见表1,所有引物序列均通过UCSC数据库比对,无已知SNP位点。
表1:PCR引物序列
表2:PCR引物序列
。
(2)试剂
本检测方法DNA提取采用酚氯仿法提取,所用试剂为PCR扩增采用Vazyme公司产品Multiplex PCR Kit,货号PM101;SNaPshot单碱基延伸测序采用ABI公司产品SNaPshot™Multiplex Kit,货号4323151;详细步骤参考各自操作说明书。
(3)主要仪器见表3
表3检测所用仪器
。
实施例2
(1)引物特异性
各引物与UCSC中进行Blasting,结果如下:VKORC1-1639 G>A扩增片段位于chr1631096202-31096491,长度为290bp;CYP4F2*3扩增片段位于chr1915879539-15879818,长度为280bp;CYP2C9*3扩增片段位于chr1094981180-94981427,长度为248bp;CYP2C19*2扩增片段位于chr1094781745-94781964,长度为220bp;CYP2C19*3扩增片段位于chr1094780531-94780850,长度为320bp;CYP2D6*10扩增片段位于chr2242130632-42130878,长度为247bp。
扩增结果如表4所示,所有引物的扩增片段均覆盖了相应的检测位点VKORC1-1639 G>A、CYP4F2*3、CYP2C9*3、CYP2C19*2、CYP2C19*3、CYP2D6*10,无其他同源片段。
表4引物扩增片段
使用表1中PCR引物分别对待测样品进行扩增及sanger测序,测序结果显示,各扩增片段与相应位点参考序列吻合,结果如图1所示;使用表2中SNaPshot PCR引物进行单碱基延伸法进行检测,结果如图2所示。由图2可知,产物峰的相对位置及测序反应参入的碱基与预期相符,无其他干扰峰。
实施例3:特异性和灵敏度检测
1、引物组合检测的特异性
本检测方法的特异性定义为阴性符合率,用SnaPshot法检测临床心内科需要使用抗凝血药物的患者样本30例,检测结果显示:VKORC1-1639 G>A阴性率为80%,CYP4F2*3阴性率为83.3%,CYP2C9*3阴性率为80%,CYP2C19*2阴性率为40%,CYP2C19*3阴性率为90%, CYP2D6* 10阴性率为33.3%。其中仅2例所有位点为阴性,与sanger测序法测试结果一致(如表5所示),本发明引物组合检测特异性为100%。
表5引物组合检测特异性
。
2、检测方法的灵敏度
本检测方法的灵敏度定义为阳性符合率,用SnaPshot法检测临床心内科需要使用抗凝血药物的患者样本30例,检测结果显示:SnaPshot 法检测到28例样本突变阳性,与sanger测序法检测结果一致(如表6所示),本发明灵敏度为100%。
表6 引物组合检测灵敏度
3、引物组合检测的准确性
本检测方法的准确性定义为不同方法检测结果一致。
本引物组合对来自昆明理工大学附属医院心内科的需要使用抗凝血药物的患者样本30例,进行检测,并与Sanger 测序法检测对比,检测结果完全一致,如表7所示;本引物组合检测的准确性为100%。
表7引物组合检测的准确性
4、精密度
本引物组合准确度定义为不同方法检测结果的一致性。
本检测进行了人员间、不同时间、同一标本不同孔间的对比实验(表8),所有结果均显示一致,本检测精密度为 100%。
表8精密度
因此,本发明所提供的引物及其组合,可作为一种独立的、应用广泛的鉴定方法,解决了抗凝血治疗相关的多种药物的基因分型鉴定问题,发挥PCR-SNaPshot对所述基因位点基因分型结果精确、高通量操作的特点,可以在临床凝血异常患者选择抗凝药物的过程中进行精确指导的作用,避免药物选择不当造成的出血或者血栓形成造成的不良事件。
以上所述仅为本发明的优选实施例而已,并不用于限制本发明,对于本领域的技术人员来说,本发明可以有各种更改和变化。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
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Claims (2)
1.一组同时检测抗凝药物相关基因多态性的引物,其特征在于:包括PCR扩增引物和SNaPshot PCR引物;同时检测的位点包括VKORC1-1639 G>A、CYP4F2*3、CYP2C9*3、CYP2C19* 2、CYP2C19*3、CYP2D6*10;
针对VKORC1-1639 G>A PCR扩增的正向引物如SEQ ID NO:1所示,反向引物如SEQ IDNO:2所示,SNaPshot PCR引物如SEQ ID NO:13所示;
针对CYP4F2*3 PCR扩增的正向引物如SEQ ID NO:3所示,反向引物如SEQ ID NO:4所示,SNaPshot PCR引物如SEQ ID NO:14所示;
针对CYP2C9*3 PCR扩增的正向引物如SEQ ID NO:5所示,反向引物如SEQ ID NO:6所示,SNaPshot PCR引物如SEQ ID NO:15所示;
针对CYP2C19*2 PCR扩增的正向引物如SEQ ID NO:7所示,反向引物如SEQ ID NO:8所示,SNaPshot PCR引物如SEQ ID NO:16所示;
针对CYP2C19*3 PCR扩增的正向引物如SEQ ID NO:9所示,反向引物如SEQ ID NO:10所示,SNaPshot PCR引物如SEQ ID NO:17所示;
针对CYP2D6*10 PCR扩增的正向引物如SEQ ID NO:11所示,反向引物如SEQ ID NO:12所示,SNaPshot PCR引物如SEQ ID NO:18所示。
2.权利要求1所述引物及其组合在临床抗凝血治疗药物选择中的应用。
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