WO2023221306A1 - 一种nppb基因dna羟甲基化标志物、引物及其应用 - Google Patents
一种nppb基因dna羟甲基化标志物、引物及其应用 Download PDFInfo
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- BNP has become a biomarker of heart failure, used for screening, diagnosis and differential diagnosis of heart failure, and is also a useful predictor of future outcomes in patients with heart failure.
- Nesiritide a recombinant human B-type natriuretic peptide synthesized based on the physiological properties of BNP, has been used clinically in patients with heart failure.
- multiple clinical studies have found that nesiritide can cause Dose-dependent hypotension and increased mortality in patients with chronic heart failure and acute renal failure. Therefore, further exploring the molecular mechanism of BNP involved in regulating cardiovascular diseases will help to develop new drugs to prevent and control cardiovascular diseases.
- the third object of the present invention is to provide the application of primers for amplifying the above-mentioned DNA hydroxymethylation markers in preparing a kit for predicting the risk of stroke.
- the upstream primer is as shown in SEQ ID NO.1
- the downstream primer is as shown in SEQ As shown in ID NO.2, specifically,
- the NPPB gene DNA hydroxymethylation marker provided by the present invention can be used to predict the risk of stroke, provide a basis for screening high-risk groups of stroke, and can also be used as an intervention target to prevent and control stroke. At the same time, detection based on multiple DNA hydroxymethylation sites overcomes the problem of low single DNA hydroxymethylation signal and improves detection sensitivity, specificity and accuracy.
- Table 2 shows the clinical characteristics of the study subjects, which included a total of 853 ischemic stroke patients (mean age 62 years, 53% male) and 918 age-sex matched healthy controls (mean age 61 years, 55% male) ). Ischemic stroke patients had more metabolic risk factors such as hypertension, diabetes, lipids, and obesity than healthy controls (P ⁇ 0.05).
- the designed primers (F: GGTTTATTTTTATATAAGGTYGGTTTTGTT; R: ACRTCCRAATTTACTTCCCACCTAC) were used to amplify the DNA of the enzyme-treated specimen to obtain an amplification product containing the T7 RNA polymerase promoter sequence.
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Abstract
本发明涉及一种NPPB基因DNA羟甲基化标志物、引物及其应用,该NPPB基因DNA羟甲基化标志物包括NPPB基因启动子区的DNA羟甲基化位点Chr1:11919160(CpG1)、Chr1:11919144(CpG2)、Chr1:11919133(CpG5)和Chr1:11919011(CpG10)中的至少一个,其羟甲基化程度指示脑卒中发病风险。本发明提供的NPPB基因DNA羟甲基化标志物可用于预测脑卒中发病风险,为预防和治疗脑卒中提供了新的方向。同时,基于上述DNA羟甲基化位点提供了一种脑卒中特异性DNA羟甲基化标志物组合,进行基于多个DNA羟甲基化位点的检测克服了单个DNA羟甲基化信号低的问题,提高了检测灵敏度、特异性和准确率。
Description
本发明涉及生物医药技术领域,尤其涉及一种NPPB基因DNA羟甲基化标志物、引物及其应用。
B型利钠肽(B-type natriuretic peptide,BNP)是利尿钠肽家族的一员,由32个氨基酸残基组成的多肽。当心室负荷增加时,心室肌细胞合成并分泌BNP参与调节心血管稳态。许多基础研究发现,BNP可通过舒张血管平滑肌—扩张血管、阻断交感神经系统—抑制肾上腺皮质激素释放、抑制肾素血管紧张素醛固酮系统—利尿排钠等过程参与调节心血管稳态以及心血管疾病及其危险因素的发生发展。在临床上,BNP已成为心衰的生物标志物,用于心衰的筛查、诊断及鉴别诊断,也是心力衰竭患者未来结局的有用预测指标。根据BNP的生理特性合成的重组人B型利钠肽——奈西立肽,已在临床上对心衰患者展开应用,然而,值得注意的是,多项临床研究发现奈西立肽会导致剂量依赖性低血压,并且会增加慢性心衰合并急性肾功能衰竭患者的病死率。因此进一步探究BNP参与调节心血管疾病的分子机制,有助于新药研发、从而预防和控制心血管疾病。左心室功能障碍、心房颤动、心力衰竭、动脉粥样硬化等疾病是已知的血浆BNP水平升高的原因,而这些心血管疾病的高发病率最终导致了中风。大量流行病学研究表明,血浆BNP水平与中风有关,入院时的血浆BNP水平可以预测中风患者的院内死亡。脑卒中是一种急性脑血管疾病,是由于脑部血管突然破裂或因血管阻塞导致血液不能流入大脑而引起脑组织损伤的一组疾病,包括缺血性和出血性卒中。脑卒中发病率和死亡率居高不下,如何有效的防治脑卒中是我国公共卫生领域面临的重大挑战。
目前,脑卒中主要的诊断方法包括颅脑CT、颅脑核磁共振、颈动脉彩超CTA和血管造影等,这些方法复杂繁琐,且影像学检查对于微小病灶的诊断能力有限,检测灵敏度和特异性低,且诊断结果具有一定的主观性。因此,临床急需新的诊断和治疗策略,探索更多的发病机制和治疗靶点将对人类健康作出重大贡献。目前尚未见关于NPPB基因DNA羟甲基化与脑卒中的研究报道,也没有报道一种合适的羟甲基化标志物可作为预防和控制脑卒中的干预靶点。
发明内容
为解决上述技术问题,本发明通过对NPPB基因DNA羟甲基化与脑卒中表型相关性的研究,提供了一种与脑卒中相关的DNA羟甲基化标志物,并进一步将其结合,提供了一种脑卒中特异性DNA羟甲基化标志物组合,进行基于多个DNA羟甲基化位点的检测克服了单个DNA羟甲基化信号低的问题,提高了检测灵敏度、特异性和准确率, 安全性也更高。
本发明的第一个目的是提供一种预测脑卒中发病风险的DNA羟甲基化标志物,该DNA羟甲基化标志物包括NPPB基因启动子区的DNA羟甲基化位点Chr1:11919160(CpG1)(CpG1)、Chr1:11919144(CpG2)(CpG2)、Chr1:11919133(CpG5)(CpG5)或Chr1:11919011(CpG10)。
进一步地,DNA羟甲基化标志物选自Chr1:11919160(CpG1)、Chr1:11919144(CpG2)、Chr1:11919133(CpG5)和Chr1:11919011(CpG10)中至少2个位点的组合。
优选地,DNA羟甲基化标志物为以下组合(1)-(10)中的任意一种:
(1)Chr1:11919160和Chr1:11919144,
(2)Chr1:11919160和Chr1:11919133,
(3)Chr1:11919160和Chr1:11919011,
(4)Chr1:11919144和Chr1:11919133,
(5)Chr1:11919144和Chr1:11919011,
(6)Chr1:11919133和Chr1:11919011,
(7)Chr1:11919160、Chr1:11919144和Chr1:11919133,
(8)Chr1:11919160、Chr1:11919144和Chr1:11919011,
(9)Chr1:11919160、Chr1:11919133和Chr1:11919011,
(10)Chr1:11919144、Chr1:11919133和Chr1:11919011,
(11)Chr1:11919160、Chr1:11919144、Chr1:11919133和Chr1:11919011。
进一步地,检测上述DNA羟甲基化标志物的羟甲基化水平包括以下步骤:
(1)提取DNA样本,用T4β-葡糖基转移酶和APOBEC3A酶处理DNA样本;
(2)扩增步骤(1)处理后的样品,得到扩增产物;
(3)对步骤(2)的扩增产物进行转录和酶切,得到转录和酶切产物;
(4)对步骤(3)的转录和酶切产物进行检测,获取序列中Chr1:11919160(CpG1)、Chr1:11919144(CpG2)、Chr1:11919133(CpG5)或Chr1:11919011(CpG10)位点的羟甲基化水平。
进一步地,DNA样本为血液样本。
进一步地,扩增DNA羟甲基化标志物的上游引物如SEQ ID NO.1所示,下游引物如SEQ ID NO.2所示。
本发明的第二个目的是提供上述DNA羟甲基化标志物在制备预测脑卒中发病风险的试剂盒中的应用。
进一步地,试剂盒中还包括扩增NPPB基因启动子区的DNA羟甲基化位点 Chr1:11919160(CpG1)、Chr1:11919144(CpG2)、Chr1:11919133(CpG5)和Chr1:11919011(CpG10)中至少一个位点的引物。
进一步地,上游引物如SEQ ID NO.1所示,下游引物如SEQ ID NO.2所示。该引物对可同时扩增出这四个位点,再分别检测每个位点的羟甲基化水平,进而判断脑卒中发病风险。
本发明的第三个目的是提供扩增上述DNA羟甲基化标志物的引物在制备预测脑卒中发病风险的试剂盒中的应用,上游引物如SEQ ID NO.1所示,下游引物如SEQ ID NO.2所示,具体地,
F:GGTTTATTTTTATATAAGGTYGGTTTTGTT
R:ACRTCCRAATTTACTTCCCACCTAC,其中,R表示碱基A/G。
本发明的第四个目的是提供一种预测脑卒中发病风险的试剂盒,该试剂盒中包括:扩增NPPB基因启动子区的DNA羟甲基化位点Chr1:11919160(CpG1)、Chr1:11919144(CpG2)、Chr1:11919133(CpG5)和Chr1:11919011(CpG10)中至少一个位点的引物。
进一步地,试剂盒中还包含检测Chr1:11919160(CpG1)、Chr1:11919144(CpG2)、Chr1:11919133(CpG5)和Chr1:11919011(CpG10)中至少一个位点羟甲基化水平的试剂,该试剂与引物对应,试剂检测的位点至少覆盖引物扩增的位点。
进一步地,上游引物如SEQ ID NO.1所示,下游引物如SEQ ID NO.2所示。
借由上述方案,本发明至少具有以下优点:
本发明提供的NPPB基因DNA羟甲基化标志物可用于预测脑卒中的发病风险,为脑卒中高危人群的筛查提供依据,也可以作为预防和控制脑卒中的干预靶点。同时,进行基于多个DNA羟甲基化位点的检测克服了单个DNA羟甲基化信号低的问题,提高了检测灵敏度、特异性和准确率。
上述说明仅是本发明技术方案的概述,为了能够更清楚了解本发明的技术手段,并可依照说明书的内容予以实施,以下以本发明的较佳实施例并配合详细附图说明如后。
为了使本发明的内容更容易被清楚的理解,下面根据本发明的具体实施例并结合附图,对本发明作进一步详细的说明。
图1为基于NGS的多目标CpG羟甲基化测序的目标序列和引物,以及预测的11个CpG位点;
图2为脑卒中患者组和健康对照组的11个CpG位点的DNA羟甲基化水平与脑卒中的关联程度;
图3-13为仅传统模型和各CpG位点联合传统模型的ROC曲线比较图;
图14-24为传统模型联合CpG位点各组合模型的ROC曲线比较图。
下面结合附图和具体实施例对本发明作进一步说明,以使本领域的技术人员可以更好地理解本发明并能予以实施,但所举实施例不作为对本发明的限定。
实施例1
在CATIS(中国急性缺血性脑卒中抗高血压试验)人群中,纳入了1771人,包括脑卒中患者853人和健康对照918人。提取每一位研究对象的全血DNA标本,运用基于NGS的多目标CpG羟甲基化测序技术检测NPPB基因启动子区域各CpG位点羟甲基化水平,即利用ENSEMBL数据库查询人类NPPB基因的启动子区域,该区域为Chr1:11919190-11918953(距TSS:-168bpto3bp),在NCBI上截取该区域的核酸序列,将核酸序列导入EMBOSSCpgplot软件预测CpG岛,然后运用Epidesigner程序对CpG岛及CpG密集区域序列进行引物设计,挑选合适的引物进行DNA羟甲基化检测(引物序列信息见表1)。经过挑选,NPPB羟甲基化共获得11个CpG位点的羟甲基化水平,11个CpG位点如图1所示。
表1 NPPB基因启动子区域DNA羟甲基化检测引物序列
DNA羟甲基化的具体检测方法为:
首先用DNA羟甲基化试剂盒对DNA标本依次进行T4β-葡萄糖基转移酶和APOBEC3A酶处理,以葡萄糖基标记样本DNA中的羟甲基化胞嘧啶,被标记了的羟甲基化胞嘧啶不会被APOBEC3A酶脱去氨基,而其余的胞嘧啶和甲基化胞嘧啶则在APOBEC3A酶的作用下转化为胸腺嘧啶。接着用表1中的引物,以酶转化后的样品基因组为模板,进行多重PCR扩增。为区分不同样品,利用带有Index序列的引物,通过PCR扩增向文库末端引入和illumina平台兼容的特异性标签序列。最终,将所有样品Index PCR扩增产物等量混合,在IlluminaHiseq/Miseq平台,以2x150bp/2x250bp的双端测序模式进行高通量测序,获得FastQ数据。各CpG位点羟甲基化水平量化为该位点羟甲基化的reads数目(即检测到碱基C的reads数目)/该位点总的reads数目×100%。
研究结果如下:
1、研究对象的临床特征
表2表示研究对象的临床特征,共包含了853名缺血性脑卒中患者(平均年龄62岁,男性占53%)和918名年龄性别匹配的健康对照(平均年龄61岁,男性占55%)。缺血性脑卒中患者相比于健康对照有更多的代谢风险因素如:高血压、糖尿病、脂质、肥胖(P<0.05)。
表2研究对象的临床特征
2、NPPB基因启动子区域DNA羟甲基化与缺血性脑卒中的关系
如表3所示,我们检测的11个CpG位点的DNA羟甲基化水平在脑卒中患者中均高于健康对照(q<0.05)。如图2所示,在调整了年龄、性别、教育水平、吸烟、饮酒、体质指数、低密度脂蛋白胆固醇、高密度脂蛋白胆固醇、高血压以及糖尿病后,CpG1(Chr1:11919160)、CpG2(Chr1:11919144)、CpG5(Chr1:11919133)、CpG9(Chr1:11919019)、CpG10(Chr1:11919011)、CpG11(Chr1:11918989)与缺血性脑卒中显著相关(P<0.05),在进行多重校正后CpG1(Chr1:11919160)、CpG2(Chr1:11919144)、CpG5(Chr1:11919133)、CpG10(Chr1:11919011)仍与脑卒中有关联(q<0.05)。
表3缺血性脑卒中患者和健康对照组的NPPB启动子羟甲基化的中位水平
为了检验CpG位点的甲基化水平能否提升传统危险因素对脑卒中的预测能力,绘制了受试者工作特征曲线(receiver operating characteristic curve ROC)并比较了曲线下面积(Area Under Curve AUC),结果如图3-13所示,并联合传统危险因素模型对脑卒中的风险预测情况进行了测定,结果见表4。如表4和图3-13所示,ROC曲线下面积:AUC(传统危险因素+CpG1羟甲基化水平)>AUC(传统危险因素+CpG2羟甲基化水平)=AUC(传统危险因素+CpG5羟甲基化水平)>AUC(传统危险因素+CpG10羟甲基化水平)>AUC(传统危险因素+CpG9羟甲基化水平)>AUC(传统危险因素+CpG4传统危险因素+CpG 4羟甲基化水平)=AUC(传统危险因素+CpG7羟甲基化水平)=AUC(传统危险因素+CpG11羟甲基化水平)>AUC(传统危险因素+CpG3羟甲基化水平)=AUC(传统危险因素+CpG6羟甲基化水平)=AUC(传统危险因素+CpG8羟甲基化水平)=AUC(仅传统危险因素);结合图2的结果(CpG1、CpG2、CpG5、CpG10在经过多重检验矫正后仍与脑卒中有关联(q<0.05)),筛选出四个位点:CpG1、CpG2、CpG5和CpG10。
表4传统模型联合各CpG位点对缺血性脑卒中的发病诊断
传统模型包括年龄、性别、教育水平、吸烟、饮酒、体质指数、低密度脂蛋白胆固醇、高密度脂蛋白胆固醇、高血压史以及糖尿病史。
如表5和图14-24所示,ROC曲线下面积(AUC)最大的模型是:传统模型+ CpG1+CpG2+CpG5+CpG10,AUC为0.866,且P值小于0.05。
表5传统模型联合CpG位点各组合模型对缺血性脑卒中的发病诊断
传统模型包括年龄、性别、教育水平、吸烟、饮酒、体质指数、低密度脂蛋白胆固醇、高密度
脂蛋白胆固醇、高血压史以及糖尿病史。
表6显示了CpG1联合CpG2、CpG5、CpG10羟甲基化水平对缺血性卒中发生风险的预测价值。在传统模型的基础上,纳入CpG1、CpG2、CpG5、CpG10羟甲基化水平后,传统模型+CpG1+CpG2+CpG5+CpG10模型对缺血性卒中发生风险的预测水平得到提升(NRI0.0475%,P<0.05,IDI0.0326%)。
表6 CpG1联合CpG2、CpG5、CpG10羟甲基化水平对缺血性卒中发生风险的预测价值
NRI:净重分类改善指数;IDI:整体鉴别指数
传统模型包括年龄、性别、教育水平、吸烟、饮酒、体质指数、低密度脂蛋白胆固醇、高
密度脂蛋白胆固醇、高血压史以及糖尿病史。
如表7所示,得出的最佳脑卒中预测模型计算公式:Logit(P)=-12.509147+0.031318*年龄+0.558676*性别+2.022532*教育水平+0.399809*吸烟+0.082706*饮酒+0.260029*体质指数-0.356943*低密度脂蛋白胆固醇+0.353775*高密度脂蛋白胆固醇+1.600649*高血压史+1.767850*糖尿病史+0.542035*CpG1羟甲基化水平+0.425184*CpG2羟甲基化水平+0.847899*CpG5羟甲基化水平+0.679995*CpG10羟甲基化水平。此模型截断值:P=0.4935596。
表7缺血性脑卒中发生情况影响因素的Logistic回归分析
Logit(P)=-12.509147+0.031318*年龄+0.558676*性别+2.022532*教育水平+0.399809*吸烟+0.082706*饮酒+0.260029*体质指数-0.356943*低密度脂蛋白胆固醇+0.353775*高密度脂蛋白胆固醇+1.600649*高血压史+1.767850*糖尿病史+0.542035*CpG1羟甲基化水平+0.425184*CpG2羟甲基化水平+0.847899*CpG5羟甲基化水平+0.679995*CpG10羟甲基化水平
实施例2构建羟甲基化检测试剂盒
基于以上研究,可以得知:位于启动子区域的CpG1(Chr1:11919160)、CpG2(Chr1:11919144)、CpG5(Chr1:11919133)和CpG10(Chr1:11919011)位点发生羟甲基化之后,可能促进NPPB基因表达进而参与脑卒中的发病,可以作为脑卒中发病风险的预测标志物和潜在药物靶点。因此,本发明构建了基于CpG1(Chr1:11919160)、CpG2(Chr1:11919144)、CpG5(Chr1:11919133)和CpG10(Chr1:11919011)位点的羟甲基化检测试剂盒。
具体检测方法为:
①全血DNA提取并质检
a.琼脂糖凝胶电泳检测基因组DNA完整性:电泳条带清晰可见,无明显降解,且无RNA污染。
b.Nanodrop2000检测基因组DNA质量:浓度≥20ng/μL,总量≥1μg,OD260/280=1.7~2.0,OD260/230≥1.8。
②T4β-葡萄糖基转移酶和APOBEC3A酶处理
用DNA羟甲基化试剂盒对质检合格的DNA标本进行T4β-葡萄糖基转移酶和APOBEC3A酶处理,将样本DNA中其余的胞嘧啶和甲基化胞嘧啶全部转化为胸腺嘧啶。
③多重PCR扩增
接着用设计好的引物(F:GGTTTATTTTTATATAAGGTYGGTTTTGTT;R:ACRTCCRAATTTACTTCCCACCTAC)对酶处理过的标本进行DNA扩增,得到带有T7RNA聚合酶启动子序列的扩增产物。
④CpG片段切割
然后运用T7RNA聚合酶将扩增的DNA产物转录为RNA片段,用RNaseA将所得的RNA片段切割成带有CpG的小片段。
⑤飞行质谱分析
最终,在每一个小的RNA片段内,未羟甲基化的CpG最终产物为CpA,羟甲基化的CpG最终产物为CpG,使用Agena MassArray飞行质谱分析系统检测这个最终产物的分子量。
⑥羟甲基化水平计算和脑卒中风险预测
该CpG位点羟甲基化水平量化为产物质量CpG/(CpG+CpA)×100%,脑卒中风险预测模型,当数据带入脑卒中预测模型:Logit(P)=-12.509147+0.031318*年龄+0.558676*性别+2.022532*教育水平+0.399809*吸烟+0.082706*饮酒+0.260029*体质指数-0.356943*低密度脂蛋白胆固醇+0.353775*高密度脂蛋白胆固醇+1.600649*高血压史+1.767850*糖尿病史+0.542035*CpG1羟甲基化水平+0.425184*CpG2羟甲基化水平+0.847899*CpG5羟甲基化水平+0.679995*CpG10羟甲基化水平,计算出预测模型P值>0.4935596时,提示脑卒中发病风险较高,应密切关注并采取预防性治疗措施。
显然,上述实施例仅仅是为清楚地说明所作的举例,并非对实施方式的限定。对于所属领域的普通技术人员来说,在上述说明的基础上还可以做出其它不同形式变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引申出的显而易见的变化或变动仍处于本发明创造的保护范围之中。
Claims (10)
- 一种预测脑卒中发病风险的DNA羟甲基化标志物,其特征在于:所述DNA羟甲基化标志物包括NPPB基因启动子区的DNA羟甲基化位点Chr1:11919160、Chr1:11919144、Chr1:11919133和Chr1:11919011中的至少一个。
- 根据权利要求1所述的DNA羟甲基化标志物,其特征在于:所述DNA羟甲基化标志物包括NPPB基因启动子区的DNA甲基化位点的组合;所述DNA甲基化位点的组合为以下组合(1)-(10)中的任意一种:(1)Chr1:11919160和Chr1:11919144,(2)Chr1:11919160和Chr1:11919133,(3)Chr1:11919160和Chr1:11919011,(4)Chr1:11919144和Chr1:11919133,(5)Chr1:11919144和Chr1:11919011,(6)Chr1:11919133和Chr1:11919011,(7)Chr1:11919160、Chr1:11919144和Chr1:11919133,(8)Chr1:11919160、Chr1:11919144和Chr1:11919011,(9)Chr1:11919160、Chr1:11919133和Chr1:11919011,(10)Chr1:11919144、Chr1:11919133和Chr1:11919011,(11)Chr1:11919160、Chr1:11919144、Chr1:11919133和Chr1:11919011。
- 根据权利要求1所述的DNA羟甲基化标志物,其特征在于,检测位点羟甲基化水平的方法包括以下步骤:(1)提取DNA样本,用T4β-葡糖基转移酶和APOBEC3A酶处理所述DNA样本;(2)扩增步骤(1)处理后的样品,得到扩增产物;(3)对步骤(2)的扩增产物进行转录和酶切,得到转录和酶切产物;(4)对步骤(3)的转录和酶切产物进行检测,获取样品序列中位点Chr1:11919160、Chr1:11919144、Chr1:11919133或Chr1:11919011的羟甲基化水平。
- 根据权利要求1所述的DNA羟甲基化标志物,其特征在于:扩增所述DNA羟甲基化标志物的上游引物如SEQ ID NO.1所示,下游引物如SEQ ID NO.2所示。
- 权利要求1-4任一项所述的DNA羟甲基化标志物在制备预测脑卒中发病风险的试剂盒中的应用。
- 扩增权利要求1-4任一项所述的DNA羟甲基化标志物的引物在制备预测脑卒中发病风险的试剂盒中的应用。
- 根据权利要求6所述的应用,其特征在于:扩增所述DNA羟甲基化标志物的上游引物如SEQ ID NO.1所示,下游引物如SEQ ID NO.2所示。
- 一种预测脑卒中发病风险的试剂盒,其特征在于,所述试剂盒中包含:扩增NPPB基因启动子区的DNA羟甲基化位点Chr1:11919160、Chr1:11919144、Chr1:11919133和Chr1:11919011中至少一个位点的引物。
- 根据权利要求8所述的试剂盒,其特征在于:所述试剂盒中还包含检测Chr1:11919160、Chr1:11919144、Chr1:11919133和Chr1:11919011中至少一个位点羟甲基化水平的试剂。
- 根据权利要求8所述的试剂盒,其特征在于:上游引物如SEQ ID NO.1所示,下游引物如SEQ ID NO.2所示。
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