CN111549043A - 一种rara相关变异型apl的融合基因及其检测引物和应用 - Google Patents
一种rara相关变异型apl的融合基因及其检测引物和应用 Download PDFInfo
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Abstract
本发明涉及临床诊断技术领域，公开了一种RARA相关变异型APL的融合基因及其检测引物和应用。本发明所述融合基因由TNRC18外显子5及其上游序列和RARA外显子3及其下游序列通过TNRC18外显子5和RARA外显子3直接融合形成。本发明提供了RARA相关APL的新融合基因TNRC18‑RARA，并针对该融合基因设计了特异性的PCR引物，扩大了原有检测手段的检测范围，能够应用于临床，可提高诊断RARA相关APL的检出率和准确率，为诊断分型及分子靶向治疗提供依据。
Description
技术领域
本发明涉及临床诊断技术领域，具体的说是涉及一种RARA相关变异型APL的融合基因及其检测引物和应用。
背景技术
急性早幼粒细胞白血病(APL)是急性白血病的一个特殊类型，发病率约占急性髓系白血病的15％。FAB协作组曾根据细胞形态学和组织化学特征将其定义为AML-M3型。鉴于异常染色体及融合基因在恶性血液肿瘤诊断、分型、微小残留病(MRD)监测及其预后中的重要价值，2008年世界卫生组织(WHO)将具有t(15；17)(q22；q21)/PML-RARa融合基因的AML直接定义为APL。约95％以上的APL可见其15号和17号染色体分别断裂并发生易位，形成t(15；17)(q22；q21)染色体异常。在基因水平上表现为15号染色体上的PML基因和l7号染色体上的维A酸受体a(RARa)基因融合，形成特征性的PML-RARa融合基因。
APL的发病机制为PML-RARA与RXRA形成二聚体，竞争野生型RARa与RXRa的结合，以阻止细胞分化，从而使骨髓中堆积大量的异常早幼粒细胞。APL患者大多在临床上起病急、凶险，常伴发弥漫性血管内凝血，从而危及生命。全反式维甲酸(ATRA)的应用，可靶向降解异常的PML-RARa融合蛋白，恢复野生型RARa基因和PML基因功能，解除对基因的转录抑制，最终使得APL细胞分化成熟。因此，ATRA的应用使得APL治疗效果得到了极大提高，而ATRA和砷剂的联合靶向治疗更使得约95％以上的APL患者可以获得长期生存甚至治愈。
目前，国内外共发现至少有14个RARa相关的伙伴基因，分别为PML、NPM1、PLZF、BCOR、FIP1L1、NuMA、PRKAR1A、OBFC2A、STAT5B、GTF2I、IRF2BP2、TBLR1、FNDC3B、STAT3。除伴有PML-RARA的典型APL外，其余称为变异型APL。虽然，这些患者具有相同的RARA,但由于其伙伴基因不同,它们对ATRA治疗的反应性不一。其中伴有PLZF-RARA、STAT5B-RARA、STAT3-RARA融合基因的APL患者对ATRA治疗不敏感或耐受，整体治疗效果不佳，预后不良，成为APL临床治疗的难题之一。
因此，APL作为一种特殊类型的白血病，近年来发现除外PML-RARa，还有许多其它RARa相关形成的融合基因，它们都具有一些相同的特征，但同时也具有一些独特的治病机理。因此，对RARA相关融合基因的精确诊断将有助于更全面的了解APL，提高该类疾病的完全缓解率，即早采取正确有效的干预措施，实施个体化治疗。
发明内容
有鉴于此，本发明的目的在于提供一种RARA相关变异型APL的新融合基因TNRC18exon5-RARA exon3，扩展了APL中RARA相关融合基因的类型；
本发明的另外一个目的在于提供上述新型融合基因TNRC18-RARA的应用；
本发明的另外一个目的在于提供上述新型融合基因TNRC18 exon5-RARA exon3的扩增引物，使其具备更高的特异性和敏感性。
为了实现上述目的，本发明提供如下技术方案：
一种RARA相关变异型APL的融合基因，所述融合基因由TNRC18外显子5及其上游序列和RARA外显子3及其下游序列通过TNRC18外显子5和RARA外显子3直接融合形成，命名为TNRC18 exon5-RARA exon3融合基因。融合后的融合基因示意图见图1。此外，也可以认为所述融合基因仅由TNRC18 exon5和RARA exon3融合形成，或所述融合基因是包含由TNRC18exon5和RARA exon3融合形成的基因的融合基因。
TNRC18基因序列在GeneBank中的登录号为NM_001080495.2，RARA基因序列在GeneBank中的登录号为NM_000964.3。其中，所述TNRC18外显子5核苷酸序列如SEQ IDNO.11所示，RARA外显子3核苷酸序列如SEQ ID NO.12所示，通过两者直接融合为TNRC18exon5-RARA exon3。
本发明通过高通量测序技术，在一例临床表现、形态学和免疫分型符合APL的初诊病例，但细胞遗传学无见t(15；17)(q22；q21)染色体异常，分子生物学未检测到PML-RARA融合基因，检测出一种新型的TNRC18-RARA融合基因，发现该基因由TNRC18外显子5和RARA外显子3融合形成，属于首次发现的变异型APL。因此，本发明提出了所述融合基因在制备以其为检测靶点的RARA相关APL的诊断试剂中的应用。
同时，本发明还提供了检测融合基因TNRC18 exon5-RARA exon3的扩增引物，一条引物以TNRC18外显子5为靶基因设计，另一条引物以RARA外显子3为靶基因设计。在本发明具体实施方式中，本发明选择TNRC18外显子5的一段序列为靶基因设计引物，序列参见SEQID NO.9；本发明选择RARA外显子3的一段序列为靶基因设计引物，序列参见SEQ ID NO.10；
针对本发明提供的新融合基因，遵循引物设计原则，引物最好在模板cDNA的保守区内设计，引物长度在15bp～30bp之间，引物GC含量在40％～60％之间，退火温度最好接近Tm值，引物自身及引物之间不应存在互补序列。在本发明具体实施方式，本发明提供的扩增引物的序列如SEQ ID NO.1和SEQ ID NO.2所示。
本发明针对不同的靶基因序列和相同引物设计原则，设计了包括SEQ ID NO.1和SEQ ID NO.2所示引物在内的4对扩增引物，扩增试验结果显示，除本发明提供的扩增引物外，其余3对扩增引物出现了存在杂带、目的条带不明显以及无法扩增出目的条带等现象；进一步利用本发明提供的扩增引物进行检测，该引物组合诊断的特异性达到了100％，敏感性达到了10-3，检测时间仅需三个工作日。
基于上述扩增引物的有益效果，本发明提出了所述扩增引物在制备检测本发明融合基因TNRC18-RARA相关APL的诊断试剂中的应用。
依据上述应用，本发明提供了一种变异型APL的诊断试剂盒，其包括以本发明融合基因TNRC18-RARA为诊断靶点的扩增引物。优选地，所述扩增引物为本发明提供的各项扩增引物技术方案。
由以上技术方案可知，本发明提供了RARA相关APL的新融合基因TNRC18-RARA，并针对该融合基因设计了特异性的PCR引物，扩大了原有检测手段的检测范围，能够应用于临床，可提高诊断RARA相关APL的检出率和准确率，为诊断分型及分子靶向治疗提供依据。
附图说明
图1所示为本发明融合基因的示意图；其中，箭头左边表示TNRC18基因外显子5及其上游序列，图中仅显示出TNRC18基因外显子1-5(ex1-5)；箭头右边表示RARA基因外显子3及其下游序列，图中仅显示出RARA基因外显子3-9(ex3-9)；
图2所示为不同引物组合的PCR扩增结果；
图3所示为扩增产物测序结果；
图4所示为TNRC18-RARA融合基因及其反向RARA-TNRC18融合基因的扩增结果；
图5所示为不同稀释度的TNRC18-RARA融合基因的扩增结果。
具体实施方式
本发明实施例公开了一种RARA相关变异型APL的融合基因及其检测引物和应用，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明内。本发明所述融合基因及其检测引物和应用已通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述融合基因及其检测引物和应用进行改动或适当变更与组合，来实现和应用本发明技术。
本发明扩增引物组合是对原有APL病例中伴随RARA融合基因检测的PCR引物的补充，扩展了APL中RARA相关融合基因的类型，增加了RT-PCR方法诊断变异型APL的检出率。
以下就本发明所提供的一种RARA相关变异型APL的融合基因及其检测引物和应用做进一步说明。
实施例1：针对明确诊断的病例进行验证和分析
患者，男性，50岁，因“突发头痛、恶心、视物模糊15小时”入苏大附一院，血常规检查显示白细胞15.54×109/L，血红蛋白75g/L，血小板62×109/L。血凝实验显示血浆凝血酶原时间(PT)为14.7sec(10.5-13.0sec)，活化部分凝血活酶时间(APTT)为24.9sec(ref.23-35sec)，纤维蛋白原水平为0.9g/L(ref.2.00-4.00g/L)，D-二聚体水平为3.830μg/mL(ref.0.00-0.55μg/mL)。骨髓形态学显示骨髓增生明显活跃，粒红比15.73:1，粒系异常增生，以病态早幼粒细胞增生为主占84.5％，该细胞大小不一，形态不规则，胞浆丰富，分内外浆，内浆布满密集噬天青颗粒，以细颗粒为主，较不均匀，核仁隐约可见。红细胞增生受抑占5.5％，全片见巨核细胞120个，形态大致正常，血片示异常早幼粒细胞占80％，成熟红细胞形态大致正常，血小板散在易见，化学染色示NAP积分值0，POX染色(+++)～(++++)。免疫分型显示CD117、CD33、CD13、CD9和MPO阳性，CD123和CD56部分阳性，CD34、HLA-DR、CD36、CD38、CD11b、CD14、CD64、CD3、CD15、CD4、CD8、CD1、CD5、CD20、cCD3、CD19、cCD79a、CD2、TDT和CD7阴性，即髓系表达。因此，其临床表现、形态学和免疫分型符合APL的诊断，初步诊断为急性早幼粒细胞白血病(APL)。
但是，该患者的染色体结果为46,XY,add(7)(p14),del(17)(q21)[2]/46,XY[5]，细胞遗传学未见t(15；17)(q22；q21)染色体异常，分子生物学未检测到PML-RARA融合基因。本发明通过高通量测序技术，检测出TNRC18-RARA融合基因，发现该基因由TNRC18外显子5和RARA外显子3融合形成。
实施例2：扩增试验
对于实施例1中高通量测序RNA-seq检测出TNRC18 exon5-RARA exon3新融合基因的病例，使用本发明设计的引物进行验证，上游引物设计的靶基因均选自SEQ ID NO.11所示核苷酸序列(即TNRC18 exon5)，下游引物设计的靶基因选自SEQ ID NO.12所示核苷酸序列(即RARA exon3)或其下游RARA exon4和RARA exon5序列和RARA exon6序列，设计原则相同，引物序列见下表1(1和2为一对引物，3和4为一对引物，以此类推)；
表1
| SEQ ID | 序列(5’——3’) |
| NO.1 | GCCCAGGGTGAGGCAGAAGT |
| NO.2 | CCCATAGTGGTAGCCTGAGGACTT |
| NO.3 | GTCGCTGCCTCCTCCTCCAA |
| NO.4 | TCGGTCGTTTCTCACAGACTCCTT |
| NO.5 | ACTCGGTCATCCGCTCGCTCAA |
| NO.6 | AGAACTGCTGCTCTGGGTCTCAAT |
| NO.7 | CCTGGTGGCTGTGGCAAGAA |
| NO.8 | GCGATGGTGAGGGTGGTGAA |
1、RNA的提取：
取患者新鲜骨髓液2mL，置于EDTA-K2抗凝管中，用细胞裂解液分离总白细胞，颠倒混匀，室温放置10分钟，1500rpm离心5分钟。采用TRIzol试剂提取标本总RNA，RNA沉淀用20μL DEPC处理水溶解。经紫外分光光度计检测RNA浓度和纯度，将RNA样本浓度调至0.5μg/mL，置于－80℃冻存。
2、逆转录PCR(RT-PCR)
按两步法操作步骤将RNA逆转录为cDNA。PCR扩增引物为表1引物组合。扩增体系包括5u/ulTaq酶0.2ul，10×Taq Buffer(含MgC12)2.5μL，10mmol/L dNTP 0.5ul(以上试剂均购自美国Promega生物公司)，10umol/L的上、下游引物各0.5ul，cDNA模板150ng，无菌去离子水加至25μL。PCR扩增条件为95℃变性10min后，94℃30s、60℃30s、72℃30s，共35次循环，最后72℃延伸7min。PCR产物用2％琼脂糖，电压100V，电泳，GelRed(购自Biotium公司)染色后于紫外光下观察结果，并送测序。
3、结果
扩增结果见图2，使用本发明引物PCR后可见单一特异的电泳条带，对扩增产物进行测序，得到TNRC18 exon5-RARA exon3融合基因融合处的序列(图3)，扩增产物的全部序列如下：
gcccagggtgaggcagaagtgcgacacccgcctgtgggcattgcagtggctgtggcccggcagaaggacagtggcggcagtggccggctggggcctgggctggtagaccaggagcgctctctgtcgctgagtaacgtcaaagCCATTGAGACCCAGAGCAGCAGTTCTGAAGAGATAGTGCCCAGCCCTCCCTCGCCACCCCCTCTACCCCGCATCTACAAGCCTTGCTTTGTCTGTCAGGACAAGTCCTCAGGCTACCACTATGGG(即SEQ ID NO.9+SEQ ID NO.10)。
而其他扩增引物中，下游引物针对的靶基因选自RARA exon4或RARA exon5，或RARA exon6，出现了条带不清、无法扩增目的条带以及出现杂带的现象，表明本发明设计的引物组合可靠且敏感，该引物可成功扩增出目的条带，条带单一、特异。同时，设计了反向融合基因RARA-TNRC18的引物，RT-PCR扩增后证明了其反向的融合基因RARA-TNRC18不存在(图4)。
实施例3：针对对照组病例进行检测
选取了30例诊断明确的典型APL病例，即这些病例除了临床表现符合APL的诊断，同时检测到PML/RAR融合基因。将这30例分别编号为C1～C30，作为对照组病例，使用本发明表1的1和2引物组合进行检测，RNA提取、逆转录PCR和测序方法同实施例2，结果见表2。
表2
同时，取实施例1病例(P1)标本与编号C1的对照组标本混合，进行倍比稀释，分别取1：10、1：100、1：1000、1：10000，共4个不同浓度点的稀释样品，使用本发明表1的1和2引物组合进行检测，RNA提取、逆转录PCR和测序方法同实施例2，结果见图4。
结果：使用本发明设计引物组合检测，30例对照组病例均未检测出TNRC18-RARA融合基因，证明本发明设计的引物特异性达100％(表2)。实施例1病例标本经稀释后，在1:1000仍能检测到明显的阳性条带，1:10000未检出，证明本发明引物组合对TNRC18-RARA融合基因的检测敏感度达10-3(图5)。
以上所述只是用于理解本发明的方法及其核心思想，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利的保护范围。
序列表
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ctttcgggcg cgagggctct ggtgcccagg gtgaggcaga agtgcgacac ccgcctgtgg 1560
gcattgcagt ggctgtggcc cggcagaagg acagtggcgg cagtggccgg ctggggcctg 1620
ggctggtaga ccaggagcgc tctctgtcgc tgagtaacgt caaag 1665
<210> 12
<211> 149
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
ccattgagac ccagagcagc agttctgaag agatagtgcc cagccctccc tcgccacccc 60
ctctaccccg catctacaag ccttgctttg tctgtcagga caagtcctca ggctaccact 120
atggggtcag cgcctgtgag ggctgcaag 149
Claims (9)
1.一种RARA相关变异型APL的融合基因，其特征在于，所述融合基因由TNRC18外显子5及其上游序列和RARA外显子3及其下游序列通过TNRC18外显子5和RARA外显子3直接融合形成。
2.权利要求1所述融合基因在制备以其为检测靶点的RARA相关APL的诊断试剂中的应用。
3.一种检测权利要求1所述融合基因的扩增引物，其特征在于，一条引物以TNRC18外显子5为靶基因设计，另一条引物以RARA外显子3为靶基因设计。
4.根据权利要求3所述扩增引物，其特征在于，所述引物在靶基因的保守区内设计，引物长度在15bp～30bp之间，引物GC含量在40％～60％之间，退火温度接近Tm值，引物自身及引物之间不应存在互补序列。
5.根据权利要求4所述扩增引物，其特征在于，所述一条引物以SEQ ID NO.9所示核苷酸序列为靶基因设计，另一条引物以SEQ ID NO.10所示核苷酸序列为靶基因设计。
6.根据权利要求3-5任意一项所述扩增引物，其特征在于，所述引物序列如SEQ IDNO.1和SEQ ID NO.2所示。
7.权利要求3-6任意一项所述扩增引物在制备检测权利要求1所述融合基因相关APL的诊断试剂中的应用。
8.一种变异型APL的诊断试剂盒，其特征在于，包括以权利要求1所述融合基因为诊断靶点的扩增引物。
9.根据权利要求8所述诊断试剂盒，其特征在于，所述扩增引物为权利要求3-6任意一项所述扩增引物。
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| CN112280866A (zh) * | 2020-11-24 | 2021-01-29 | 福州艾迪康医学检验所有限公司 | 荧光定量pcr技术筛查急性早幼粒细胞白血病相关14种融合基因的方法、引物及探针 |
| CN114350804A (zh) * | 2022-01-07 | 2022-04-15 | 中南大学湘雅二医院 | 一种aml相关的融合基因及其应用，检测引物、试剂盒 |
| CN117165610A (zh) * | 2023-10-31 | 2023-12-05 | 南昌大学第一附属医院 | 一种rara相关变异型apl的融合基因及其扩增引物 |
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