CN116656816A - 一种zfand3-tfeb融合基因及其检测引物和应用 - Google Patents
一种zfand3-tfeb融合基因及其检测引物和应用 Download PDFInfo
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Abstract
本发明公开了一种ZFAND3‑TFEB融合基因及其检测引物和应用。一种ZFAND3‑TFEB融合基因，其特征在于基因融合发生于ZFAND3的1号外显子与TFEB基因2号外显子之间，包含SEQ ID NO.3所示的核苷酸序列。本发明克隆了一个TFEB的新易位伴侣，为ZFAND3‑TFEB基因易位，在TFEB易位性肾细胞癌中为首次报道，本发明针对该新融合位点设计了特异性的PCR引物，扩大了原引物组合的检测范围，应用于临床，可提高诊断该类肿瘤的检出率和准确率。
Description
技术领域
本发明属于医学检验领域，涉及一种ZFAND3-TFEB融合基因及其检测引物和应用。
背景技术
2016年WHO对2004年肾细胞癌病理组织学分类进行了修改，新增了TFEB基因易位相关性肾癌(TFEB易位性肾癌)。TFEB基因位于6p21.1，是TFEB易位性肾癌的唯一驱动基因，其致病机理清晰明确：这类肿瘤均涉及位于6p21.1的TFEB基因同其它染色体易位及其所致形成融合基因，通过启动子变换从而高表达TFEB融合蛋白，而TFEB作为一个转录因子，通过与特异的DNA结构相结合，转录调控体内多种基因表达最终致病。TFEB易位性肾癌最常见的易位形式为MALAT1-TFEB，近年来散在个案报道指出，TFEB易位性肾癌存在不同的易位伴侣和融合基因，包括KHDRBS2-TFEB,COL21A1-TFEB,CADM2-TFEB等。申请人多年致力于TFEB易位性肾癌的研究，发现了多种导致TFEB易位性肾癌的融合基因，如CN201710458099.3中公开的MATR3的15号外显子与TFE3基因4号外显子之间发生基因融合而成MATR3-TFE3融合基因，CN201910641043.0中公开的发生于EWSR1的6号外显子与TFEB基因5号外显子之间的融合基因EWSR1-TFEB融合基因，等等。但是由于该疾病以发病年龄轻为突出特征，因此造成极其沉重的家庭及社会负担，因此有待发掘更多的致病基因，及检测这些新的致病基因的检测试剂。
发明内容
本发明的目的是针对现有技术的上述不足，提供一种TFEB的新易位伴侣。
本发明的另一目的是提供该新易位伴侣的检测引物。
本发明的又一目的是提供引物的应用。
本发明的目的可通过以下技术方案实现：
一种TFEB的新易位伴侣，为ZFAND3-TFEB基因易位，ZFAND3基因位于6p21.2(6号染色体短臂2区1带2亚带，位置37787503-38122400，序列来自GeneBank，序列版本号GRCh37/hg19)，共7个外显子；TFEB基因位于6p21.1(6号染色体，位置41,651,716-41,703,997，序列来自GeneBank，序列版本号GRCh37/hg19),共17个外显子。基因融合发生于ZFAND3的1号外显子与TFEB基因2号外显子之间。我们通过高通量测序方法检测未知融合对象的TFEB肿瘤患者的标本，发现所述的TFEB的新易位伴侣：ZFAND3-TFEB基因易位，包含SEQ ID NO.3所示的核苷酸序列。在本专利所述TFEB易位性肾细胞癌中，这一易位对象国内外尚未见报道。
本发明所述的ZFAND3-TFEB融合基因(或称ZFAND3-TFEB基因易位)的检测试剂在制备ZFAND3-TFEB易位性肾细胞癌诊断试剂中的应用。
所述的ZFAND3-TFEB融合基因的检测试剂为检测ZFAND3-TFEB融合基因的引物组合物。所有能够检测本发明所述ZFAND3-TFEB基因易位的引物对均在本发明的保护范围内；优选上游引物ZFAND3-E1-F如SEQ ID NO.1所示，下游引物TFEB-E2-R如SEQ ID NO.2所示。
一种用于诊断ZFAND3-TFEB易位性肾细胞癌的引物组合物，为检测本发明所述的ZFAND3-TFEB基因易位的PCR引物；优选上游引物ZFAND3-E1-F如SEQ ID NO.1所示，下游引物TFEB-E2-R如SEQ ID NO.2所示。
针对本发明找到的新的新易位伴侣，我们在ZFAND3的1号外显子和TFEB的2号外显子内分别设计引物。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72℃，引物自身及引物之间不应存在互补序列，扩增条带单一特异。由于工作中经常需要在石蜡固定标本中开展工作，石蜡标本RNA碎裂严重，因此扩增产物不宜过长，需要控制在350bp以下。
经过多次调试和验证，最终设计的引物序列为：ZFAND3-E1-F：TCGCTGTCCCTGCGGCTTCT；(SEQ ID NO.1)；TFEB-E2-R:CTGCTGCTGCTGCTGCATGTAAT(SEQ IDNO.2)，理论产物长度163bp，扩增产物(融合基因)的全部序列如下：TCGCTGTCCCTGCGGCTTCTGGGGGGAGCCAGCGCCGGC AGCCACCATGGCGTCACGCATAGGGTTGCGCATGCAGCTCATGCGGGAGCAGGCGCAGCAGGAGGAGCAGCGGGAGCGCAT GCAGCAACAGGCTGTCATGCATTACATGCAGCAGCAGCAGCAG(SEQ IDNO.3)。
经实验验证，引物可成功扩增出目的条带，条带单一、特异(图1)。验证病例实际扩增产物长度163bp，融合基因序列如下:TCGCTGTCCCTGCGGCTTCTGGGGGGAGCCAGCGCCGGCAGCCACCAT GGCGTCACGCATAGGGTTGCGCATGCAGCTCATGCGGGAGCAGGCGCAGCAGGAGGAGCAGCGGGAGCGCATGCAGCAACA GGCTGTCATGCATTACATGCAGCAGCAGCAGCAG(SEQ ID NO.3)，使用本发明引物可扩增出融合基因序列。
本发明所述的引物组合物在制备ZFAND3-TFEB易位性肾细胞癌诊断试剂中的应用。
一种ZFAND3-TFEB易位性肾细胞癌诊断试剂盒，包含本发明所述的引物组合物。
有益效果：
本发明克隆了一个TFEB的新易位伴侣，为ZFAND3-TFEB基因易位，在TFEB易位性肾细胞癌中为首次报道，且该基因易位为同一染色体臂内易位，无法被荧光原位杂交方法检出。本发明针对高通量测序发现的新融合位点设计了特异性的PCR引物，扩大了原引物组合的检测范围，应用于临床，可提高诊断该类肿瘤的检出率和准确率。为诊断分型及分子靶向治疗提供依据。根据我们的实验结果，该引物组合诊断的特异性和敏感性均达到了100％，且操作对象只需要在石蜡包埋组织切片上进行，时间仅为三个工作日。采用本发明提供的探针组合进行检测ZFAND3-TFEB易位性肿瘤，不但方便、快速、可靠而且检出率高，可用于制备ZFAND3-TFEB易位性肿瘤诊断试剂盒，为ZFAND3-TFEB易位性肿瘤的快速准确的诊断提供了新的工具。
附图说明
图1：在已知ZFAND3-TFEB融合型肿瘤中应用本发明引物组合(ZFAND3-E1-F；TFEB-E2-R)进行验证，成功检出的ZFAND3-TFEB融合基因测序图。
图2：对上述已知ZFAND3-TFEB融合型肿瘤送检高通量测序(RNA-Seq)验证，测出融合基因序列与本专利设计引物所扩增出的融合基因序列一致。
图3：在已知ZFAND3-TFEB融合型肿瘤中应用本发明引物组合(ZFAND3-E1-F；TFEB-E2-R)进行验证，逆转录PCR后的电泳条带图，于163bp左右可见单一特异条带(8号孔)。对照组病例使用本发明引物组合(ZFAND3-E1-F；TFEB-E2-R)进行逆转录PCR，扩增后电泳条带图，均未扩增出目标条带(1-7号孔)。
具体实施方式
以下通过实施例对本发明作进一步的阐述。
实施例1针对明确诊断的病例进行验证：
对于高通量测序RNA-seq检测出ZFAND3 exon1-TFEB exon2新融合位点的病例，(患者为江苏泰州人，30岁女性，于2022年5月腹部CT检查发现肾脏占位，至中国人民解放军东部战区总医院就诊，腹腔镜下行左肾切除术，术后病理检查疑为TFEB基因易位易位性肾细胞癌，但免疫组化TFEB蛋白阳性与荧光原位杂交TFEB基因断裂探针阴性出现不一致结果，最终通过高通量测序RNA-seq确诊存在ZFAND3-TFEB基因易位)使用我们设计的引物进行验证。
一、RNA的提取：
严格按照RNeasy FFPE Kit操作说明进行提取。①脱蜡：将收集好的玻片进行二甲苯脱蜡，再用无水乙醇漂洗，风干后用手术刀片刮下来装入1.5ml EP管中；②酶解：加入150μl消化液,再加入10μl蛋白酶K，混匀，56℃酶解15min,80℃15min，冰上冷却；③加入16μlDNDNA酶缓冲液,再加入10μl DNase I,混匀，室温静置15mimin,12000rpm离心15min，取上清；④加入320μl结合液液再加入720μl无水乙醇，混匀，分2次转移至吸附柱中，8000rpm离心1min，，弃废液；⑤洗涤：加入500μl洗涤液,8000rpm离心1min；重复洗涤一次，弃废液，将吸附柱转移到一个新的2ml收集管中，12000rpm离心5min；⑥洗脱：将吸附柱转移到1.5ml的EP管中，加入100μl的洗脱液，室温静置1min,12000rpm离心1min，将收集好的洗脱液(即DNA提取液)进行浓度和纯度测定，-80℃保存存待用。
二、逆转录PCR RT-PCR
采用试剂盒(K1622，RevertAid First Strand cDNA Synthesis Kit，MBI)对RNA进行逆转录，方法详见试剂盒说明。PCR扩增引物为本专利ZFAND3-TFEB融合基因引物组合ZFAND3-E1-F/TFEB-E2-R。反应体系包括：0.125μl TaKaRa Ex TaqTMHS液，2.5μl 10×TaqBuffer(Mg2+plus)，2μl dNTP(均购自日本Takara公司)，引物浓度为20μmol/l，cDNA模版为100ng，无菌去离子水加至25μl。PCR扩增条件为94℃变性3min后，94℃30s、60℃30s、72℃1min，共35次循环，最后72℃延伸5min。PCR产物用3％琼脂糖，电压100V，电泳，溴化乙啶染色后于紫外光下观察结果，并送测序。
结果：分别用本发明引物(ZFAND3-E1-F/TFEB-E2-R)PCR后可见单一特异的约163bp的电泳条带(图3中8号孔)，对扩增产物进行测序，得到ZFAND3 exon1-TFEB exon2融合基因序列(图1)，证明本发明设计的引物组合可靠且敏感。
实施例2针对对照组病例进行检测
我们对30例诊断明确的对照组病例(包括10例透明细胞性肾细胞癌，5例乳头状肾细胞癌，5例嫌色细胞性肾细胞癌，5例TFE3易位相关性肾细胞癌和5例MALAT1-TFEB易位相关性肾癌，理论上均不存在ZFAND3-TFEB基因融合)使用本发明的引物组合进行检测，RNA提取、逆转录PCR和测序方法同上。
结果：使用本发明设计引物组合检测，未检测出ZFAND3-TFEB融合基因，PCR后未见电泳条带(图3中1-7号孔)，证明本项目设计的引物特异性高。
评价：本发明引物组合是对原有TFEB易位性肾细胞癌融合基因引物的补充，扩展了TFEB易位性肾细胞癌融合基因的类型，增加了RT-PCR方法诊断该肿瘤的检出率。
Claims (9)
1.一种ZFAND3-TFEB融合基因，其特征在于基因融合发生于ZFAND3的1号外显子与TFEB基因2号外显子之间，包含SEQ ID NO.3所示的核苷酸序列。
2.权利要求1所述的ZFAND3-TFEB融合基因在制备ZFAND3-TFEB易位性肾细胞癌诊断试剂中的应用。
3.权利要求1所述的ZFAND3-TFEB融合基因的检测试剂在制备ZFAND3-TFEB易位性肾细胞癌诊断试剂中的应用。
4.根据权利要求3所述的应用，其特征在于所述的ZFAND3-TFEB融合基因的检测试剂为检测ZFAND3-TFEB融合基因的引物组合物。
5.根据权利要求4所述的应用，其特征在于所述ZFAND3-TFEB基因易位的引物组合物由SEQ ID NO.1所示的上游引物ZFAND3-E1-F和SEQ ID NO.2所示的下游引物TFEB-E2-R组成。
6.一种用于诊断ZFAND3-TFEB易位性肾细胞癌的引物组合物，其特征在于为检测权利要求1所述的ZFAND3-TFEB基因易位的PCR引物。
7.根据权利要求6所述的引物组合物，其特征在于由SEQ ID NO.1所示的上游引物ZFAND3-E1-F和SEQ ID NO.2所示的下游引物TFEB-E2-R组成。
8.一种检测权利要求1所述的ZFAND3-TFEB融合基因所致ZFAND3-TFEB易位性肾细胞癌的诊断试剂盒，其特征在于含有检测权利要求1所述的ZFAND3-TFEB基因易位的PCR引物。
9.根据权利要求8所述的诊断试剂盒，其特征在于检测权利要求1所述的ZFAND3-TFEB基因易位的PCR引物由SEQ ID NO.1所示的上游引物ZFAND3-E1-F和SEQ ID NO.2所示的下游引物TFEB-E2-R组成。
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