CN114752672B - 基于循环游离DNA突变进行滤泡性淋巴瘤预后评估的检测panel、试剂盒及应用 - Google Patents
基于循环游离DNA突变进行滤泡性淋巴瘤预后评估的检测panel、试剂盒及应用 Download PDFInfo
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Abstract
本发明公开了一种基于循环游离DNA突变进行滤泡性淋巴瘤预后评估的检测panel、试剂盒及应用。所述检测panel包括检测滤泡性淋巴瘤预后相关的突变基因。本专利提供了滤泡性淋巴瘤的预后评估的非侵入性的检测方法,具有临床应用价值,以便更好地指导治疗、改善FL患者的预后。创造性地选择了与滤泡性淋巴瘤密切相关的基因组合,基于二代测序技术,检测和分析内容包括:对FL临床患者的血浆cfDNA(plasma cfDNA),石蜡淋巴瘤组织DNA(tumor tissue gDNA)以及粒细胞DNA(granulocyte gDNA)进行二代检测,本发明的基因组合对预后评估、治疗有指导意义,为医生提供更多的预后评估维度。
Description
技术领域
本发明属于滤泡性淋巴瘤基因突变检测技术领域,具体涉及一种基于循环游离DNA突变进行滤泡性淋巴瘤预后评估的检测panel、试剂盒及应用。
背景技术
疗效和预后评价在肿瘤治疗中具有重要的临床意义,其评价方式和方法多种多样。目前,滤泡性淋巴瘤的诊断和预后评估需要组织学、免疫组织化学(如CD19、CD20染色)和分子遗传学分析,这些分析是通过侵入性程序(如活检)获得的活检组织和肿瘤组织切片。然而,通过侵入性方法获得的活检有几个缺点:
(1)患者可能会感到疼痛和其他与侵入性活检相关并发症;
(2)肿瘤活检可能难以获得,重复取样难度极大;
(3)因为肿瘤的异质性,整个肿瘤组织的遗传全貌无法获得;
(4)不能动态监测滤泡性淋巴瘤免疫化疗治疗的反应。目前用于滤泡性淋巴瘤诊断和分期的非侵入性方法(如PET、PET-CT)价格昂贵,也不能排除假阳性结果,影像学检查并不能代替淋巴瘤组织活检。
因此,随着临床和实验研究的进步,分子诊断技术的提高,对FL预后的预测方法也需要不断完善和发展,以便更好地指导治疗、改善FL患者的预后。
发明内容
本发明的目的是要解决上述的技术问题,本发明提供了一种基于循环游离DNA突变进行滤泡性淋巴瘤预后评估的检测panel、试剂盒及应用。
为了解决上述问题,本发明按以下技术方案予以实现的:
第一方面,本发明提供了一种基于循环游离DNA突变进行滤泡性淋巴瘤预后评估的检测panel,所述检测panel包括检测滤泡性淋巴瘤预后相关的突变基因。
结合第一方面,本发明还提供了第一方面的第1种优选实施方式,具体的,所述检测panel的突变基因由110个基因组成,所述基因的名称列举如下:
ABCC10;AGTPBP1;ARHGEF1;ARID1A;ATP6AP1;ATP6V1B2;AUTS2;B2M;BAZ2B;BCL10;BCL2;BCL6;BCL7A;BRWD3;BTG1;C4orf49;CALR;CARD11;CASC5;CASZ1;CCAR1;CCND3;CCNK;CD40;CD79A;CD79B;CEP112;CHRM3;CPNE1;CREBBP;CTSS;DIRAS3;DMRT3;DTX1;EBF1;EEF1A1;ENPP3;ENTPD4;EP300;ERBB2;ETV1;EZH2;FAT2;FN1;FOXO1;GNA13;GNAI2;GNE;GRM7;HIST1H1B;HIST1H1C;HIST1H1D;HIST1H1E;HIST1H2AC;HIST1H2AG;HIST1H2BC;HIST1H2BD;HIST1H2BG;IKZF2;IKZF3;IL13RA1;IRF8;KIR2DL3;KLHDC7B;KLHL6;KMT2C;KMT2D;KNDC1;LAMP1;LPHN1;LYST;MATN2;MCL1;MEF2B;MON2;MUC4;MYD88;NEB;NOTCH2;NOTCH1;POU2F2;OR2M3;PCLO;PEX14;PLCG2;PRKCB;RBM23;ROS1;SLC9A6;SOCS1;SP140;STAT3;STAT6;SVIL;TLR1;TNFAIP3;TNFRSF14;TP53;TRAFD1;UPF1;USP6;VPS39;WDR64;ZCCHC6;ZNF141;ZNF236;ZNF423;ZNF541;ZNF595;ZNF672。
结合第一方面,本发明还提供了第一方面的第2种优选实施方式,具体的,所述检测panel还包括用于对滤泡性淋巴瘤相关基因突变进行交叉验证的引物,所述引物用于对110个基因中高于20%的体细胞突变位点进行PCR-Sanger测序验证。
结合第一方面,本发明还提供了第一方面的第3种优选实施方式,具体的,所述引物包括:
(1)检测CREBBP基因突变的引物:上游引物SEQ ID NO:1;下游引物SEQ ID NO:2。
(2)检测HIST1H1E基因突变的引物:上游引物SEQ ID NO:3;下游引物SEQ ID NO:4。
(3)检测ARID1A基因突变的引物:上游引物SEQ ID NO:5;下游引物SEQ ID NO:6。
(4)检测STAT6基因突变的引物:上游引物SEQ ID NO:7;下游引物SEQ ID NO:8。
(5)检测CTSS基因突变的引物:上游引物SEQ ID NO:9;下游引物SEQ ID NO:10。
(6)检测HIST1H1E基因突变的引物:上游引物SEQ ID NO:11;下游引物SEQ ID NO:12。
(7)用于检测ATP6AP1基因突变的引物:上游引物SEQ ID NO:13;下游引物SEQ IDNO:14。
(8)用于检测IRF8基因突变的引物:上游引物SEQ ID NO:15;下游引物SEQ ID NO:16。
(9)用于检测KMT2D基因突变的引物:上游引物SEQ ID NO:17;下游引物SEQ IDNO:18。
第二方面,本发明还提供了一种预后评估滤泡性淋巴瘤的试剂盒,所述试剂盒包括探针panel和/或检测引物,所述探针panel为针对第一方面所述的突变基因。
第三方面,本发明还提供了一种预后评估滤泡性淋巴瘤的试剂盒,所述试剂盒包括探针panel和/或检测引物;所述探针panel用于检测第一方面所述的与滤泡性淋巴瘤预后相关的突变基因;所述检测引物为第一方面所述的用于对突变基因进行交叉验证的引物。
第四方面,本发明还提供一种根据第一方面所述的检测panel在制备滤泡性淋巴瘤医学产品中的应用,所述滤泡性淋巴瘤医学产品包括用于预后评估滤泡性淋巴瘤。
第五方面,本发明还提供了基于循环游离DNA中的基因组合在预后评估滤泡性淋巴瘤的应用,所述的基因组合由如下110个基因组成:ABCC10;AGTPBP1;ARHGEF1;ARID1A;ATP6AP1;ATP6V1B2;AUTS2;B2M;BAZ2B;BCL10;BCL2;BCL6;BCL7A;BRWD3;BTG1;C4orf49;CALR;CARD11;CASC5;CASZ1;CCAR1;CCND3;CCNK;CD40;CD79A;CD79B;CEP112;CHRM3;CPNE1;CREBBP;CTSS;DIRAS3;DMRT3;DTX1;EBF1;EEF1A1;ENPP3;ENTPD4;EP300;ERBB2;ETV1;EZH2;FAT2;FN1;FOXO1;GNA13;GNAI2;GNE;GRM7;HIST1H1B;HIST1H1C;HIST1H1D;HIST1H1E;HIST1H2AC;HIST1H2AG;HIST1H2BC;HIST1H2BD;HIST1H2BG;IKZF2;IKZF3;IL13RA1;IRF8;KIR2DL3;KLHDC7B;KLHL6;KMT2C;KMT2D;KNDC1;LAMP1;LPHN1;LYST;MATN2;MCL1;MEF2B;MON2;MUC4;MYD88;NEB;NOTCH2;NOTCH1;POU2F2;OR2M3;PCLO;PEX14;PLCG2;PRKCB;RBM23;ROS1;SLC9A6;SOCS1;SP140;STAT3;STAT6;SVIL;TLR1;TNFAIP3;TNFRSF14;TP53;TRAFD1;UPF1;USP6;VPS39;WDR64;ZCCHC6;ZNF141;ZNF236;ZNF423;ZNF541;ZNF595;ZNF672。
结合第五方面,本发明还提供了第五发明的第1种优选实施方式,具体的,在所述的应用中,将所述的基因组合作为二代测序系统的检测基因集,通过二代测序系统检测临床患者的血浆cfDNA、肿瘤组织DNA和/或外周血粒细胞DNA中基因组合的突变情况,并根据基因组合的突变情况预后评估滤泡性淋巴瘤。
结合第五方面,本发明还提供了第五发明的第2种优选实施方式,具体的,所述的预后评估具体为:
(1)当从血浆cfDNA和/或肿瘤组织DNA中检测到BCL2基因突变,FL患者的OS和PFS较短;
(2)当从肿瘤组织DNA中检测到CCND3基因突变,FL患者的OS和PFS较短;
(3)当从血浆cfDNA中检测到BCL2基因突变,且FL患者的Ki-67指数、LDH水平高或PET/CT阳性区域数较高时,FL患者的OS和PDS最差。
与现有技术相比,本发明的有益效果是:
1、本专利提供了滤泡性淋巴瘤的预后评估的非侵入性的检测方法,具有临床应用价值,以便更好地指导治疗、改善FL患者的预后。
2、本发明创造性地选择了与滤泡性淋巴瘤密切相关的基因组合,基于二代测序技术,检测和分析内容包括:对FL临床患者的血浆cfDNA(plasma cfDNA),石蜡淋巴瘤组织DNA(tumor tissue gDNA)以及粒细胞DNA(granulocyte gDNA)进行二代检测,检测FL临床患者的体细胞突变及其与临床病理指标的关系,旨在达到精准检测。本发明的基因组合对预后评估、治疗有指导意义,为医生提供更多的预后评估维度。
附图说明
下面结合附图对本发明的具体实施方式作进一步详细的说明,其中:
图1是本发明的FL患者血浆cfDNA和肿瘤组织体细胞突变统计;
图2是本发明的cfDNA检测到的体细胞突变基因统计;
图3是本发明的临床病理指标和FL患者预后相关图示;
图4是本发明的血浆cfDNA中BCL2突变预示FL患者预后不良图示;
图5是本发明的cfDNA存在BCL2突变和不良临床病理指标的FL患者生存率的图示;
图6是本发明的一例FL患者cfDNA中存在早期进展相关基因突变的图示;
图7是本发明的PCR-Sanger测序ARID1A R1722*交叉验证图示。
具体实施方式
以下结合附图对本发明的优选实施例进行说明,应当理解,此处所描述的优选实施例仅用于说明和解释本发明,并不用于限定本发明。
目前,滤泡性淋巴瘤的诊断和预后评估需要组织学、免疫组织化学(如CD19、CD20染色)和分子遗传学分析,这些分析是通过侵入性程序(如活检)获得的活检组织和肿瘤组织切片。然而,通过侵入性方法获得的活检存在诸多弊端。
因此,本发明提供了对FL预后的液体活检的预测方法,具体为提供110个基因组合以用于二代测序检测FL淋巴瘤cfDNA突变及其与临床病理指标的关系,特别是与患者早期进展(即在确诊24个月内发生病情进展progression of disease within 24months,POD24)发生的关系。同时,还提供了9个基因组合及引物序列采用PCR-Sanger测序检测cfDNA突变的技术体系,以便更好地指导治疗、改善FL患者的预后。
本发明实施例提供了一种基于循环游离DNA突变进行滤泡性淋巴瘤预后评估的检测panel,所述检测panel包括检测滤泡性淋巴瘤预后相关的突变基因。其中,所述检测panel的突变基因由110个基因组成,所述基因的名称列举如下:
ABCC10;AGTPBP1;ARHGEF1;ARID1A;ATP6AP1;ATP6V1B2;AUTS2;B2M;BAZ2B;BCL10;BCL2;BCL6;BCL7A;BRWD3;BTG1;C4orf49;CALR;CARD11;CASC5;CASZ1;CCAR1;CCND3;CCNK;CD40;CD79A;CD79B;CEP112;CHRM3;CPNE1;CREBBP;CTSS;DIRAS3;DMRT3;DTX1;EBF1;EEF1A1;ENPP3;ENTPD4;EP300;ERBB2;ETV1;EZH2;FAT2;FN1;FOXO1;GNA13;GNAI2;GNE;GRM7;HIST1H1B;HIST1H1C;HIST1H1D;HIST1H1E;HIST1H2AC;HIST1H2AG;HIST1H2BC;HIST1H2BD;HIST1H2BG;IKZF2;IKZF3;IL13RA1;IRF8;KIR2DL3;KLHDC7B;KLHL6;KMT2C;KMT2D;KNDC1;LAMP1;LPHN1;LYST;MATN2;MCL1;MEF2B;MON2;MUC4;MYD88;NEB;NOTCH2;NOTCH1;POU2F2;OR2M3;PCLO;PEX14;PLCG2;PRKCB;RBM23;ROS1;SLC9A6;SOCS1;SP140;STAT3;STAT6;SVIL;TLR1;TNFAIP3;TNFRSF14;TP53;TRAFD1;UPF1;USP6;VPS39;WDR64;ZCCHC6;ZNF141;ZNF236;ZNF423;ZNF541;ZNF595;ZNF672。
在本发明的优选实施中,所述检测panel还包括用于对突变基因进行交叉验证的引物,所述引物用于对110个基因中高于20%的体细胞突变位点进行PCR-Sanger测序验证。所述引物如表1所示。
表1 PCR-Sanger测序进行交叉验证PCR引物
本发明的相关实验如下:
(1)定制110个基因的二代测序基因集:本发明将FL及发生组织转化的FL(transformed FL,tFL)基因突变,创造性地定制了包含上述110个基因的二代测序检测基因集。
(2)选择13例有症状和7例无症状FL患者的临床病理特征(表2),并提取了20名患者血浆cfDNA(plasma cfDNA),石蜡淋巴瘤组织DNA(tumor tissue gDNA)以及粒细胞DNA(granulocyte gDNA),采用包含上述二代测序基因集的二代测序系统进行检测。
表2. 20例FL患者的临床病理特征
(3)研究发现:FL患者血浆cfDNA存在肿瘤组织来源的体细胞突变,有症状FL患者血浆cfDNA比无症状者含有更高比例的体细胞突变:
图1中,(A)所有FL患者血浆cfDNA和肿瘤组织DNA中检测到的体细胞突变数量;(B)血浆cfDNA和肿瘤组织DNA样本中检测到的等位基因变异分数(variant allelefractions,VAFs)之间的Spearman相关性分析;有症状(C)和无症状(D)FL患者血浆cfDNA和肿瘤组织DNA中检测到的体细胞突变数量和百分比;(E)所有患者和有症状、无症状患者体细胞突变数;(F)每个有症状和无症状FL患者的体细胞突变数。(G)在13例有症状和7例无症状FL病例中检测到的体细胞突变包括错义(missense)、无义(nonsense)、插入缺失(indel)和剪接体突变(splice site)。
通过研究发现,对20名FL患者进行检测基因集的110个FL相关基因中,共检出31个基因存在91个体细胞突变,30个体细胞突变(33%)在血浆cfDNA和肿瘤组织DNA均可检测到,而56个体细胞突变(61.5%)和5个体细胞突变(5.5%)分别只在肿瘤组织DNA或血浆cfDNA中检测到(图1A);更为重要的是,肿瘤组织和血浆cfDNA的体细胞突变的等位基因变异分数(variant allele fractions,VAFs)存在显著正相关(R=0.53,p=0.002)(图1B),进一步支持了cfDNA中检测到的体细胞突变来源于患者肿瘤组织。
而在91个体细胞突变中的65个存在于有症状FL患者,26个存在于无症状FL患者;65个在有症状FL患者中检测到的体细胞突变,27个(41.5%)在cfDNA和肿瘤组织DNA中均可检测到,34个(52.3%)仅在肿瘤组织DNA中检测到,4个(6.2%)仅在cfDNA中检测到(图1C);26个在无症状FL患者中检测到的体细胞突变,3个(11.6%)在cfDNA和肿瘤组织DNA中均可检测到,22个(84.6%)仅在肿瘤组织DNA中检测到,1个(3.8%)仅在cfDNA中检测到(图1D)。在有症状的FL患者中,血浆cfDNA存在的体细胞突变比例较高;而在无症状患者中,检测到的大多数突变存在于肿瘤组织DNA中(图1E)。更为重要的是,我们在13例有症状的FL病例中的10例(77%)和7例无症状病例中的1例(14.3%)的肿瘤组织DNA和血浆cfDNA中至少同时存在1个突变,每个有症状FL患者的突变数量略高于无症状患者(图1F)。65个在有症状FL患者中检测到体细胞突变,其中54个错义突变(missense)、6个无义突变(nonsense)、2个插入缺失突变(indel)和3个剪接体突变(splice site);26个在无症状FL患者中检测到体细胞突变,其中17个错义突变、6个无义突变、2个插入缺失突变以及1个剪接体突变(图1G)。
(4)研究结果表明与FL发病机制相关的基因突变存在于cfDNA:
如图2所示的瀑布图直观反应突变频率,在20例FL病例中,高频突变基因有:CREBBP(40%)、BCL2(30%)、STAT6(25%)、CARD11(20%)和EZH2(20%)(图2A);CREBBP突变通常位于HAT/KAT结构域,表明酶活性可能发生功能性变化(图2B);BCL2突变分布在蛋白质的不同部位,而STAT6突变富集在STAT结合域,EZH2突变主要在SET域(图2B);在5种STAT6错义突变中,其中3种(即p.D419N、p.D419N和p.D523V)同时存在于FL患者的cfDNA和肿瘤组织DNA中;此外,所有STAT6突变的患者也携带CREBBP突变,与新近的研究结果一致【BloodCancer J,2020,10(6):69】。在4名FL患者中鉴定出6种EZH2突变,其中2种突变同时存在于cfDNA和肿瘤组织DNA;EZH2突变中有4种位于热点突变Y646。
(5)临床研究表明在治疗前的体细胞突变,在接受免疫化疗后消失:
通过观察有症状的FL患者在接受免疫化疗后,体细胞突变是否持续存在,并比较了化疗前和化疗后血浆cfDNA样本中VAFs的百分比变化。12例接受免疫化疗的FL患者中,有9例在化疗前血浆cfDNA样本中至少存在1个体细胞突变,而在治疗后血浆cfDNA样本中,在30个错义、无义、插入缺失突变中仅检测到1个插入缺失突变(即CARD11 p.S622DEL)。
(6)研究发现临床病理指标与FL预后的关系:
如图3所示,PFS生存分析显示Ki-67指数(A)、血清LDH水平(B)和PET/CT阳性区域数(C)与FL患者PFS显著相关。本发明研究了Ki-67指数、血清LDH水平、PET-CT阳性区域数与FL预后的关系,结果表明Ki-67高表达(p=0.013)、血清LDH水平高(p=0.00076)和PET-CT阳性区域数多(p=0.00014)的FL患者PFS显著缩短(图3)。
(7)研究发现BCL2突变的FL患者OS及PFS缩短:
如图4所示,本发明通过评估31个突变基因与FL患者的OS和PFS的关系,生存分析表明cfDNA和/或肿瘤组织DNA检测到BCL2基因突变,患者的OS较短(p=0.0042,图4A),PFS有缩短的趋势,但无统计学差异(p=0.054,图4D);类似地,血浆cfDNA的BCL2突变也提示较短的OS(p=0.00074,图4B)及PFS(p=0.0072,图4E);还观察到CCND3突变的FL患者OS(p<0.0001,图4C)及PFS(p=0.0072,图4F)明显缩短,然而,CCND3突变仅在肿瘤组织存在。其他突变基因与FL患者的OS和PFS无关。
此外,cfDNA携带BCL2突变且Ki-67指数高(图5A)、LDH水平高(图5B)或PET/CT阳性区域数高(图5C)的FL患者OS最差;同样,cfDNA携带BCL2突变且Ki-67指数高(图5D)、LDH水平高(图5E)或PET/CT阳性区域数高(图5F)的FL患者PFS最差。
(8)研究表明cfDNA检测到早期进展和组织学转化相关基因突变:
如图6所示,FL患者血浆cfDNA样本检测到早期进展和组织学转化相关的BTG1p.E46D(A)和B2M p.Q22*(B)突变,而患者匹配的肿瘤组织DNA及对照白细胞DNA未检测到突变。(C)患者治疗前后的PET/CT显像。
研究表明BTG1和B2M等基因突变可以识别发生早期进展和组织学转化的FL患者【PLoS Med,2016,13(12):e1002197】。本发明发现1例早期进展FL患者血浆cfDNA中存在BTG1 p.E46D(图6A)和B2M p.Q22*(图6B)突变,而肿瘤组织DNA及对照白细胞DNA均没有检出BTG1和B2M突变,PET-CT显像显示R-CHOP免疫化疗6个月后完全缓解,免疫化疗12个月出现进展(图6C)。
(10)PCR-Sanger测序对突变基因进行交叉验证:
本发明使用表1的9个基因突变进行PCR-Sanger测序验证,并采用9个基因的PCR验证引物组合引物。结果与二代测序结果100%符合(表3),展示其中PCR-Sanger交叉验证的ARID1Ap.R1722*突变结果(图7)。图7中,血浆cfDNA(A)、肿瘤组织DNA(B)、对照白细胞DNA(C)中ARID1A R1722*无义突变分别通过二代测序(左图)和PCR-Sanger测序检测(右图)。
表3 PCR-Sanger测序交叉验证结果
AA:Amino acid氨基酸
NGS:Next generation sequencing二代测序
VAF:Variant allele fraction等位基因变异分数
FFPE tDNA:Formalin-fixed paraffin-embedded tumor tissue DNA石蜡固定肿瘤组织DNA
Granulocyte:Granulocyte gDNA白细胞DNA
基于本发明记载的相关试验,本发明提供了一种预后评估滤泡性淋巴瘤的试剂盒,所述试剂盒包括探针panel和/或检测引物,所述探针panel为针对上述110个突变基因的组合。
本发明还提供了一种预后评估滤泡性淋巴瘤的试剂盒,其特征在于,所述试剂盒包括探针panel和/或检测引物;所述探针panel用于检测上述110个与滤泡性淋巴瘤预后相关的突变基因;所述检测引物为表1中用于对突变基因进行交叉验证的引物。
本发明还提供了一种将上述检测panel在制备滤泡性淋巴瘤医学产品中的应用,所述滤泡性淋巴瘤医学产品包括用于预后评估滤泡性淋巴瘤。
本发明还提供一种基于循环游离DNA中的基因组合在预后评估滤泡性淋巴瘤的应用,所述的基因组合由如上述的110个基因组成。
在一种优选实施中,在所述的应用中,将所述的基因组合作为二代测序系统的检测基因集,通过二代测序系统检测临床患者的血浆cfDNA、肿瘤组织DNA和/或外周血粒细胞DNA中基因组合的突变情况,并根据基因组合的突变情况预后评估滤泡性淋巴瘤。
基于(7)研究发现BCL2突变的FL患者OS及PFS缩短,本发明还提供了预后评估具体为:
(1)当从血浆cfDNA和/或肿瘤组织DNA中检测到BCL2基因突变,FL患者的OS和PFS较短;
(2)当从肿瘤组织DNA中检测到CCND3基因突变,FL患者的OS和PFS较短;
(3)当从血浆cfDNA中检测到BCL2基因突变,且FL患者的Ki-67指数、LDH水平高或PET/CT阳性区域数较高时,FL患者的OS和PDS最差。
结合上述的预后评估的应用,本发明还提供了一种滤泡性淋巴瘤预后评估的检测方法,包括以下步骤:
步骤100:样品DNA质量检测(Evaluation of DNA Quality)
获得检测样本DNA(所述样本DNA包括血液样本的DNA组织样本DNA及cfDNA),提取病人的病灶组织及癌旁或血液组织进行DNA提取,石蜡包埋组织亦可。
其中,高质量的基因组DNA是进行基因组测序实验的前提。基因组DNA质量检测通过以下两种方法进行:(1)琼脂糖凝胶电泳检测基因组DNA完整性:要求电泳条带清晰可见,无明显拖尾;(2)Nanodrop 2000/Qubit检测基因组DNA浓度及质量:要求浓度≥50ng/μL,总量≥1.5μg,OD260/280=1.8~2.0。
步骤200:分选纯化DNA及片段化(DNA Purification and Shearing)
取基因组DNA,使用Covaris进行片段化。片段化后,DNA长度大部分介于100-500bp之间。
步骤300:末端修复反应(End Repair)
样本DNA文库的构建,将DNA随机片段化成几百碱基或更短的小片段,进行文库制备。主要步骤有:DNA打断,末端补平,3’端加“A”,接头连接,纯化,文库扩增,纯化。
片段化后的DNA存在5’或3’端突出,向纯化后的DNA片段中加入末端补齐体系,其中T4 DNA聚合酶(T4 DNA Polymerase)的外切酶(Exonuclease)活性消化3’端的单链突出,而聚合酶(Polymerase)活性补齐5’端的突出;同时磷酸激酶(PNK)在5’末端加上后续连接反应必需的磷酸基团,经过Agencourt AMpure XP磁珠纯化,最终得到5’端含有磷酸基团的平末端DNA短片段文库。
向上述体系中加入3’末端加“A”缓冲反应体系。在末端修饰完成的双链DNA3’末端加上单个腺苷酸“A”,防止DNA片段之间的平末端自连,还可以与下一步测序接头5’末端的单个“T”突出互补配对,准确连接,有效降低文库片段之间自身的串联。
步骤400:连接测序接头(Adapter Ligation)
向上述反应体系中加入连接缓冲液和双链测序接头,利用T4 DNA连接酶将illumina测序接头连接至文库DNA两端。
步骤500:文库片段筛选(Size Selection)
对于加上接头的文库,应用Agencourt SPRIselect核酸片段筛选试剂盒在纯化文库的同时,进行片段大小筛选。采用两步法筛选(Double Size selection),先用SPRI磁珠去掉目标区域左侧小片段(Left-side Size selection),再去掉位于目标片段区域右侧的大片段(Right-side Size selection)最终筛选出片段长度适中的原始文库,用于下一步的PCR扩增。经过纯化后的文库,去掉了体系中过量的测序接头和接头自连的产物,避免PCR过程的无效扩增,消除对上机测序的影响。
步骤600:PCR扩增DNA文库(PCR Amplification)
应用高保真的聚合酶扩增原始文库,以保证足够的文库总量。此外因为只有两端都连有接头的DNA片段才能够被扩增,因此该步骤还能够有效富集这部分DNA。PCR扩增循环数控制在10-12之间。在保证产物足够的前提下,减少因扩增循环数过大而引入的bias。最终使用Qubit精确测定每个文库浓度。
步骤700:芯片杂交(Library Hybridization with Array)
使用Roche NimbleGen特异化芯片试剂盒,文库和探针杂交,进行目的区域捕获。
步骤800:杂交文库清洗及纯化(Library Wash and Purification)
借助探针上偶联的生物素基团,利用MyOneTMStreptavidin T1磁珠富集探针。经步骤700)中杂交步骤后,和探针特异性结合的文库DNA一同被富集。在经过进一步清洗去除和磁珠非特异性结合的DNA后,文库中属于目的区域的DNA得到富集。
步骤900:PCR扩增DNA文库(PCR Amplification)
在50μL反应体系中应用高保真的聚合酶扩增原始文库,以保证足够的文库总量。PCR扩增循环数控制在10-12之间。在保证产物足够的前提下,减少因扩增循环数过大而引入的bias。扩增后的文库经过磁珠纯化即成为可以上机的测序文库。
步骤1000:文库的质量检测(Library Quality Assessment)
使用Qubit和Agilent 2100Bioanalyzer分别检测文库浓度与文库片段长度分布。要求浓度>5ng/μl,且片段长度集中在300-400bp之间。
步骤1100:上机测序(Sequencing)
文库最终于Illumina Hiseq平台,以2×150bp双端测序模式进行高通量测序,获得FastQ数据。
步骤1200:分析步骤
靶向目标区域测序生物信息分析主要流程:1)使用TrimGalore对原始数据过滤,然后使用软件FLASH将经过滤的R1、R2 reads拼接。并用FastX处理拼接获得的fastq文件,得到fa格式序列;2)过滤后的reads使用BWA进行基因组比对;3)使用GATK的标准流程进行SNVs calling和Indels calling。主要步骤包括,去除重复reads,对测序碱基质量值(basequality score)进行校正,以患者对应的外周粒细胞作为正常对照,使用GATK的Mutect2模块对每个患者整合进行SNVs calling和Indels calling,用多个标准对初步得到的SNVs和Indels进行过滤,得到最终的SNVs和Indels数据集。根据基因突变情形,对患者进行预后评估。
以上所述,仅是本发明的较佳实施例而已,并非对本发明作任何形式上的限制,故凡是未脱离本发明技术方案内容,依据本发明的技术实质对以上实施例所作的任何修改、等同变化与修饰,均仍属于本发明技术方案的范围内。
SEQUENCE LISTING
<110> 广州医科大学附属肿瘤医院
<120> 基于循环游离DNA突变进行滤泡性淋巴瘤预后评估的检测panel、试剂盒及应
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Claims (6)
1.基于循环游离DNA突变进行滤泡性淋巴瘤预后评估的检测panel,其特征在于,所述检测panel包括检测滤泡性淋巴瘤预后相关的突变基因;
其中,所述检测panel的突变基因由110个基因组成,所述基因的名称列举如下:
ABCC10;AGTPBP1;ARHGEF1;ARID1A;ATP6AP1;ATP6V1B2;AUTS2;B2M;BAZ2B;BCL10;BCL2;BCL6;BCL7A;BRWD3;BTG1;C4orf49;CALR;CARD11;CASC5;CASZ1;CCAR1;CCND3;CCNK;CD40;CD79A;CD79B;CEP112;CHRM3;CPNE1;CREBBP;CTSS;DIRAS3;DMRT3;DTX1;EBF1;EEF1A1;ENPP3;ENTPD4;EP300;ERBB2;ETV1;EZH2;FAT2;FN1;FOXO1;GNA13;GNAI2;GNE;GRM7;HIST1H1B;HIST1H1C;HIST1H1D;HIST1H1E;HIST1H2AC;HIST1H2AG;HIST1H2BC;HIST1H2BD;HIST1H2BG;IKZF2;IKZF3;IL13RA1;IRF8;KIR2DL3;KLHDC7B;KLHL6;KMT2C;KMT2D;KNDC1;LAMP1;LPHN1;LYST;MATN2;MCL1;MEF2B;MON2;MUC4;MYD88;NEB;NOTCH2;NOTCH1;POU2F2;OR2M3;PCLO;PEX14;PLCG2;PRKCB;RBM23;ROS1;SLC9A6;SOCS1;SP140;STAT3;STAT6;SVIL;TLR1;TNFAIP3;TNFRSF14;TP53;TRAFD1;UPF1;USP6;VPS39;WDR64;ZCCHC6;ZNF141;ZNF236;ZNF423;ZNF541;ZNF595;ZNF672;
所述检测panel用于检测滤泡性淋巴瘤预后相关的突变基因信息,根据突变基因信息和预后指标,确定滤泡性淋巴瘤的预后状态;
所述预后指标包括BCL2基因突变信息、CCND3基因突变信息、BCL2基因突变信息和Ki-67指数高表达的组合、BCL2基因突变信息和高LDH水平的组合、以及BCL2基因突变信息和高PET/CT阳性区域数的组合;
所述预后状态包括预后差状态和预后极差状态,所述预后差状态对应的预后指标为BCL2基因突变信息或CCND3基因突变信息;所述预后极差状态对应的预后指标为BCL2基因突变信息和Ki-67指数高表达的组合、BCL2基因突变信息和高LDH水平的组合、以及BCL2基因突变信息和高PET/CT阳性区域数的组合。
2.根据权利要求1所述的基于循环游离DNA突变进行滤泡性淋巴瘤预后评估的检测panel,其特征在于:
所述检测panel还包括用于对滤泡性淋巴瘤相关基因突变进行交叉验证的引物,所述引物用于对110个基因中高于20%的体细胞突变位点进行PCR-Sanger测序验证。
3.根据权利要求2所述的基于循环游离DNA突变进行滤泡性淋巴瘤预后评估的检测panel,其特征在于,所述引物包括:
(1)用于检测CREBBP基因突变的引物:
上游引物SEQ ID NO:1对应的核苷酸序列为5'-AAATGACAGGACGGTACTTACG-3';
下游引物SEQ ID NO:2对应的核苷酸序列为5'-GGACCAGTTCACCCAAGTATG-3';
(2)用于检测HIST1H1E基因突变的引物:
上游引物SEQ ID NO:3对应的核苷酸序列为5'-GGGAAGCCAAGCCTAAG-3';
下游引物SEQ ID NO:4对应的核苷酸序列为5'-GGCTTCTTCGCCTTCTTT-3';
(3)用于检测ARID1A基因突变的引物:
上游引物SEQ ID NO:5对应的核苷酸序列为5'-AATTCTGTTCTTAGGCCACTTT-3';
下游引物SEQ ID NO:6对应的核苷酸序列为5'-CACCCACCTCATACTCCTTTA-3';
(4)用于检测STAT6基因突变的引物:
上游引物SEQ ID NO:7对应的核苷酸序列为5'-TATGGCTGCTCAGACTACC-3';
下游引物SEQ ID NO:8对应的核苷酸序列为5'-CCCTAGGAGATCCTGCTG-3';
(5)用于检测CTSS基因突变的引物:
上游引物SEQ ID NO:9对应的核苷酸序列为5'-ACTAAGCATTTAAAGAGCTCTACCT-3';
下游引物SEQ ID NO:10对应的核苷酸序列为5'-TTGCCTGATTCTGTGGACTG-3';
(6)用于检测HIST1H1E基因突变的引物:
上游引物SEQ ID NO:11对应的核苷酸序列为5'-GCTCATTACTAAAGCTGTTGCC-3';
下游引物SEQ ID NO:12对应的核苷酸序列为5'-TGTTGTTCTTCTCCACGTCAT-3';
(7)用于检测ATP6AP1基因突变的引物:
上游引物SEQ ID NO:13对应的核苷酸序列为5'-CCTCGAAGTCCACAGCAAT-3';
下游引物SEQ ID NO:14对应的核苷酸序列为5'-ACGAGGAGACTACCCTTCTT-3';
(8)用于检测IRF8基因突变的引物:
上游引物SEQ ID NO:15对应的核苷酸序列为5'-TCTGTCTTTCCAAGGATGTGTG-3';
下游引物SEQ ID NO:16对应的核苷酸序列为5'-CAGCGTGTTTCCAAGGGAT-3';
(9)用于检测KMT2D基因突变的引物:
上游引物SEQ ID NO:17对应的核苷酸序列为5'-TGCAGCTGTTTCCTTCTCC-3';
下游引物SEQ ID NO:18对应的核苷酸序列为5'-CCCTATATCGCTCCTGTCTCT-3'。
4.一种预后评估滤泡性淋巴瘤的试剂盒,其特征在于,所述试剂盒包括探针panel和/或检测引物,所述探针panel为针对权利要求1所述的突变基因。
5.一种预后评估滤泡性淋巴瘤的试剂盒,其特征在于,所述试剂盒包括探针panel和/或检测引物;所述探针panel用于检测权利要求2或3中所述的与滤泡性淋巴瘤预后相关的突变基因;所述检测引物为权利要求2或3中所述的用于对突变基因进行交叉验证的引物。
6.一种根据权利要求1至3任意一项所述的检测panel在制备滤泡性淋巴瘤医学产品中的应用,所述滤泡性淋巴瘤医学产品包括用于预后评估滤泡性淋巴瘤。
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