CN116287204A - 检测特征基因的突变情况在制备静脉血栓栓塞症风险检测产品中的应用 - Google Patents
检测特征基因的突变情况在制备静脉血栓栓塞症风险检测产品中的应用 Download PDFInfo
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Abstract
本发明提供了一种检测特征基因的突变情况在制备静脉血栓栓塞症风险检测产品中的应用,所述特征基因包括FABP2。本发明基于静脉血栓栓塞散发人群基因组数据分析得到特征基因及SNP位点;首次筛选得到与基于多基因突变特征的VTE致病相关联的FABP2特征基因,为人群频率低的致病相关的罕见突变基因的获得和应用开拓了新的发展前景,为制备静脉血栓栓塞症风险检测产品提供了更多的可能性和更好的技术支撑。
Description
技术领域
本发明属于基因检测领域,具体涉及检测特征基因的突变情况在制备静脉血栓栓塞症风险检测产品中的应用。
背景技术
静脉血栓栓塞症(Venous thromboembolism,VTE)是指血液在静脉内不正常地凝结导致血管阻塞从而引起静脉回流障碍,是世界第三大循环系统致死性疾病,仅次于心肌梗死和卒中。VTE包括肺血栓栓塞症(Pulmonary thromboembolism,PTE)和下肢深静脉血栓形成(Deep venous thrombosis,DVT)两种,它们是VTE在不同部位、不同阶段的两种重要的临床表现形式。VTE在不同种族间的发病率有明显差异,VTE的发病率亚洲人群较西方人群低。
VTE是一种多因素引起的复杂疾病,病因主要分为获得性因素及遗传性因素两方面。研究表明VTE的遗传学因素在不同种族间差异明显,西方国家人群静脉血栓的常见的突变风险因素主要是导致促凝因素增强的FVLeiden突变和凝血酶原G20210A突变。这两个位点的突变在亚洲人群中非常少见,几乎没有研究报道。在中国,常见的VTE易感基因主要是蛋白C、蛋白S、THBD以及SERPINC1等。最近的研究对全球VTE家系进行了系统地荟萃分析,汇总了287个有明确遗传致病因素的VTE家系,包含了225个高加索家系,39个中国家系,以及其他散在的家系。发现这5个基因的突变最高:F5,SERPINC1,PROC,F2,PROS1,但F2和F5的突变在高加索家系中常见,在中国家系中相对少见。
随着测序技术的发展,全基因组测序(WGS)和全外显子组测序(WES)提供了全基因水平的突变筛查,更容易发现新的突变位点。欧洲最大规模的家系研究是在2016年,分析了212个VTE核心家系的GWAS以及24对家系中兄弟姐妹的靶向测序数据,发现了SMAP1、B3GAT2和RIMS1三个潜在的致病基因。2019年,Blood期刊发表的综述文献,提到了38个已知VTE相关的基因。但对于因人群频率低的部分致病的罕见突变,现有技术仍然难以发现。
发明内容
为此,本发明所要解决的技术问题在于提供检测特征基因的突变情况在制备静脉血栓栓塞症风险检测产品中的应用。解决现有技术中对于与基于多基因突变特征的静脉血栓栓塞症中致病相关联的罕见突变难以发现的技术问题。
本发明提供的一个技术方案为检测特征基因的突变情况在制备静脉血栓栓塞症风险检测产品中的应用,所述特征基因包括FABP2。
优选的,所述特征基因还包括OR6S1、LOC101929470、ZNF469、SP6、TMEM132D、RBFOX1、DNAH17、MAGI1、VKORC1L1、ZNF808、ABCC4、LINC01075、RPS6KC1中的一个或多个。
优选的,所述特征基因还包括FGG和/或ABO。
优选的,所述产品包括用于检测静脉血栓栓塞症风险的试剂、试剂盒、芯片、高通量测序平台、风险预测模型或风险评估系统。
优选的,所述风险预测模型基于研究样本的基因组数据进行分析并筛选出具有预测价值的SNP位点,得到与VTE是否患病显著相关的特征基因,以所述特征基因作为特征参数,搭建回归模型,进行训练与测试;所述研究样本由VTE患病者样本和健康者样本组成,所述VTE患病者样本为无诱因的散发静脉血栓栓塞病例。
优选的,所述与VTE是否患病显著相关的特征基因信息如下:
优选的,所述与VTE是否患病显著相关的特征基因的筛选方法包括如下步骤:
S1、收集研究样本及基因检测实验;
S2、数据质量控制及全基因组关联分析(GWAS);
S3、筛选具有预测价值的SNP位点组合;
S4、验证具有预测价值的SNP位点。
优选的,FABP2的转录活性在FABP2-A163G突变下提高。优选的,FABP2的蛋白表达水平在FABP2-A163G突变下提高。
优选的,突变情况包括可变剪切突变、移码插入突变、移码删除突变、无义突变、错义突变、非移码插入突变和非移码删除突变。
有益效果:
本发明提供的检测特征基因的突变水平在制备静脉血栓栓塞症风险检测产品中的应用,基于散发人群基因组数据分析得到特征基因及SNP位点;首次筛选得到与基于多基因突变特征的VTE致病相关联的FABP2特征基因,为人群频率低的致病相关的罕见突变基因的获得和应用开拓了新的发展前景,为制备静脉血栓栓塞症风险检测产品提供了更多的可能性和更好的技术支撑。
本发明的方法得出风险预测模型可以更加精确的评估患者的静脉血栓的遗传风险。尤其适用于中国人。通过本发明基于多基因突变特征的静脉血栓栓塞症风险评估模型预测VTE患病风险,更加准确的反应VTE风险,以便及时采取预防措施、降低发病风险和死亡风险,对中国人VTE的预防具有重要意义。
附图说明
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为本发明研究样本的队列示意图;
图2为本发明全基因组关联分析(GWAS)结果;
图3为本发明VTE风险评估模型在散发人群中的效果;
图4为本发明基础实验验证具有预测价值的SNP位点的分析结果。
具体实施方式
为详细说明本发明的技术内容、所实现目的及效果,以下结合实施方式予以说明。
本文中的英文缩写含义及英文名词解释:
GWAS:全基因组关联分析;
rsID:the rs number in the dbSNP database,dbSNP数据库中的rs编号;
MAF:Minor allele frequency in public database,公共数据库中等位基因频率较低;
OR:odds ratio.优势比,是分析疾病与因素之间联系程度的指标;
CI:confidence interval,置信区间;
AUC:Area Under The Curve,曲线下面积。
本发明的具体实施方式提供了一种检测特征基因的突变情况在制备静脉血栓栓塞症风险检测产品中的应用。其中,特征基因包括FABP2,进一步包括OR6S1、LOC101929470、ZNF469、SP6、TMEM132D、RBFOX1、DNAH17、MAGI1、VKORC1L1、ZNF808、ABCC4、LINC01075、RPS6KC1中的一个或多个;更进一步包括FGG和/或ABO。突变情况包括七种基因突变类型,具体为:可变剪切突变、移码插入/删除突变、无义突变、错义突变、非移码插入/删除突变;产品包括用于检测静脉血栓栓塞症风险的试剂、试剂盒、芯片、高通量测序平台、风险预测模型或风险评估系统。
实施例1
本实施例提供一种与基于多基因突变特征的静脉血栓栓塞症显著相关的特征基因的筛选方法,包括如下步骤:
S1、收集研究样本及基因检测实验
研究样本包括VTE患病者样本和健康者样本。
本实施例的研究样本选自中国汉族18931个样本,其中1268例无诱因的散发静脉血栓栓塞病例为患病者样本,17663名健康对照为健康者样本。
其中,散发人群研究队列的入组条件是,根据临床指南确诊静脉血栓栓塞,该临床研究经中日友好医院机构评审委员会批准(No.2016-SSW-7),收集所有入组VTE患者的临床信息和外周血样本,提取DNA冻存,所有获取的信息都进行了隐私保护。
本发明健康者样本来自深圳微基因公司数据库。所有个人参与者均已进行在线研究知情同意。该研究得到深圳市微基因临床实验室伦理委员会的批准。
对研究样本进行基因芯片测序,分别获得了1268例患者和17663例对照组的基因芯片测序数据。
S2、数据质量控制及GWAS
本实施例中主要使用PLINK v1.9软件进GWAS。先将原始数据FASTQ文件式转换,基因分型,比对到人类参考基因组GRCh37/hg19上,生成原始的VCF文件。再经过结果数据预处理。包括去除亲缘关系2级以内的样本;根据千人基因组数据库匹配,去除非汉族人群祖源的样本;筛除最小等位基因频率值(Minor allel frequence,MAF)计算<0.01的SNP位点;筛除性别基因信息与临床信息不匹配样本;筛除SNP丢失率>0.05位点的样本;筛除哈迪温伯格(Hardy—Weinberg Equilibrium,HWE)P值<1×10-6的SNP位点。
参见图1,将研究样本随机分为两组,选取样本数量(N)9475例作为筛选阶段的训练集,另外样本数量(N)9456例作为验证阶段的测试集,将特征选择过程与模型测试过程相互独立,进行GWAS分析。通过GWAS分析,得出FABP2、FGG、OR6S1、LOC101929470、ZNF469、SP6、TMEM132D、RBFOX1、ABO、DNAH17、MAGI1、VKORC1L1、ZNF808、ABCC4、LINC01075、RPS6KC1达到全基因组关联分析的显著差异(5×10-8)。
S3、筛选具有预测价值的SNP位点
全基因显著性检验针对所有位点进行计算。得到每个基因的p value、风险程度OR值(Odd Ratio),OR值大于1表示基因突变数量越多、VTE疾病的危险度增加,两者呈正相关。按p value依次对全基因进行排序,参见表1,得到与VTE是否患病显著相关的特征基因及SNP位点信息。
表1 16个特征基因及SNP位点信息
其中,rsID表示SNP位点编号;Chromosome Position表示所在染色体上的位置。
S4、基础实验验证具有预测价值的SNP位点
S41、FABP2-A163G促进FABP2基因转录;
FABP2-A163G促进FABP2基因转录。为探究FABP2转录活性,将PEGFP-N1,FABP2-WT,FABP2-A163G质粒转染进HEK293T细胞中,qPCR结果表明,与FABP2-WT相比,FABP2-A163G突变促进FABP2基因转录,参见图4。
S42、FABP2-A163G突变促进FABP2蛋白表达;
为进一步研究FABP2蛋白表达的影响,收集转染EGFP-N1,FABP2-WT,FABP2-A163G质粒的HEK293T细胞全细胞裂解液。Western Blots结果表明,与FABP2-WT相比,FABP2-A163G突变显著促进FABP2蛋白表达,参见图4。
实施例2
本实施例提供与基于多基因突变特征的静脉血栓栓塞症显著相关的特征基因在制备静脉血栓栓塞症风险检测产品中的组合方式。
第一种,特征基因中包含FABP2。
第二种,基于第一种组合,特征基因中还包含OR6S1、LOC101929470、ZNF469、SP6、TMEM132D、RBFOX1、DNAH17、MAGI1、VKORC1L1、ZNF808、ABCC4、LINC01075、RPS6KC1中的一个或多个。
第三种,基于第二种组合,特征基因中还包含FGG和/或ABO。
基于以上特征基因的组合形式,用于制备静脉血栓栓塞症风险检测产品,包括用于检测静脉血栓栓塞症风险的试剂、试剂盒、芯片、高通量测序平台、风险预测模型或风险评估系统。
其中,所述试剂产品包括特异性识别上述特征基因的探针,或特异性扩增上述特征基因的引物。
进一步的,所述试剂盒产品包含上述试剂。
实施例3
本实施例提供一种基于多基因突变特征的静脉血栓栓塞症风险预测模型,基于实施例1的筛选方法得到显著相关的特征基因,基于实施例2的特征基因组合方式,搭建回归模型,进行训练与测试。构建方法包括如下步骤:
S1、收集研究样本及基因检测实验;
S2、数据质量控制及全基因组关联分析(GWAS);
S3、筛选具有预测价值的SNP位点组合;
S4、验证具有预测价值的SNP位点;
S5、得到与VTE是否患病显著相关的特征基因;
S6、搭建回归模型,进行训练与测试。
本实施例根据所有罕见突变位点计算的VTE显著相关基因排名,按照两组排名结果分别选取靠前的基因作为特征参数进行逻辑回归建模,得到VTE风险预测模型。
针对288个SNP位点、16个特征基因的风险预测模型,对每个样本进行计算赋值后,提示患病者中评分在整体分布5%及95%的人群较健康对照组存在显著差异,AUC可达0.765,参见图3。
本实施例构建的VTE风险预测模型,能够更加精确的预测VTE患病风险,相比于现有技术风险预测模型具有更高的准确率,本模型更适合亚洲人群,特别是在中国人群中进行VTE的风险筛查,进而辅助医务人员识别出临床上存在的高危患者,进行必要的预防与干预措施,降低VTE的发生率和死亡率,提升医疗质量。
实施例4
本实施例提供了基于实施例3提供的风险预测模型在临床中对患者进行VTE风险预测,包括如下步骤:
(1)采集该患者的外周静脉血液,对血液进行抗凝操作;
(2)从采集的外周血中提取基因组DNA,并进行质检和浓度测定;
(3)质检合格后,进行遗传易感位点检测,检测方法可以使用基因芯片或二代测序,检测内容基于实施例2中特征基因的组合方式,本实施例采用第(3)种组合方式中的16个基因组合;
(4)根据实施例3构建的模型,各项特征的回归系数,计算风险分数。
根据风险分数可以得知该患者的VTE易感风险,继而根据患病风险及时采取相应的预防措施。
显然,上述实施例仅仅是为清楚地说明所作的举例,而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的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸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)
1.检测特征基因的突变情况在制备静脉血栓栓塞症风险检测产品中的应用,其特征在于,所述特征基因包括FABP2。
2.根据权利要求1所述的应用,其特征在于,所述特征基因还包括OR6S1、LOC101929470、ZNF469、SP6、TMEM132D、RBFOX1、DNAH17、MAGI 1、VKORC1 L1、ZNF808、ABCC4、LINC01075、RPS6KC1中的一个或多个。
3.根据权利要求1或2所述的应用,其特征在于,所述特征基因还包括FGG和/或ABO。
4.根据权利要求1所述的应用,其特征在于,所述产品包括用于检测静脉血栓栓塞症风险的试剂、试剂盒、芯片、高通量测序平台、风险预测模型或风险评估系统。
5.根据权利要求4所述的应用,其特征在于,所述风险预测模型基于研究样本的基因组数据进行分析并筛选出具有预测价值的SNP位点,得到与VTE是否患病显著相关的特征基因,以所述特征基因作为特征参数,搭建回归模型,进行训练与测试;所述研究样本由VTE患病者样本和健康者样本组成,所述VTE患病者样本为无诱因的散发静脉血栓栓塞病例。
7.根据权利要求5或6所述的应用,其特征在于,所述与VTE是否患病显著相关的特征基因的筛选方法包括如下步骤:
S1、收集研究样本及基因检测实验;
S2、数据质量控制及全基因组关联分析(GWAS);
S3、筛选具有预测价值的SNP位点组合;
S4、验证具有预测价值的SNP位点。
8.根据权利要求1所述的应用,其特征在于,FABP2的转录活性在FABP2-A163G突变下提高。
9.根据权利要求1所述的应用,其特征在于,FABP2的蛋白表达水平在FABP2-A163G突变下提高。
10.根据权利要求1所述的应用,其特征在于,所述突变情况包括可变剪切突变、移码插入突变、移码删除突变、无义突变、错义突变、非移码插入突变和非移码删除突变。
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CN117789819A (zh) * | 2024-02-27 | 2024-03-29 | 北京携云启源科技有限公司 | Vte风险评估模型的构建方法 |
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