CN114107452A - 一种基于fbn1基因插入突变的马凡综合征检测试剂盒 - Google Patents
一种基于fbn1基因插入突变的马凡综合征检测试剂盒 Download PDFInfo
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- CN114107452A CN114107452A CN202111482761.1A CN202111482761A CN114107452A CN 114107452 A CN114107452 A CN 114107452A CN 202111482761 A CN202111482761 A CN 202111482761A CN 114107452 A CN114107452 A CN 114107452A
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Abstract
本发明提供了一种基于FBN1基因c.5688‑5689插入突变的马凡综合征检测试剂盒,属于分子生物检测技术领域。本发明通过对马凡综合征家族成员的全血DNA进行碱基序列分析,在家族患病成员中检测到了FBN1基因的c.5688‑5689insG碱基插入变异(p.Arg1897Glufs),家系中的未患病成员均未检测到该变异。通过检测受试者是否携带FBN1基因c.5688‑5689插入突变,可以检测该变异的携带者,有助于马凡综合征的分子诊断和家系遗传分析,同时可以研究基因突变与临床表型之间的关系,为其将来可能需要的产前诊断提供依据,同时为马凡综合征的药物研发提供新的靶点。
Description
技术领域
本发明涉及分子生物检测技术领域,具体涉及一种基于FBN1基因插入突变的马凡综合征检测试剂盒,尤其是涉及一种基于FBN1基因c.5688-5689插入突变的马凡综合征检测试剂盒。
背景技术
马凡综合征(Marfan syndrome)是一种常染色体显性、全身性结缔组织疾病,发病率为2/10000-3/10000。马凡综合征常累计心血管、眼部以及骨骼等多个系统。患者临床主要表现为身材瘦长、蜘蛛样指、脊柱侧凸、漏斗胸或鸡胸,眼部晶状体异位或脱位、高度近视、视网膜脱离以及心脏主动脉扩张、主动脉夹层、二尖瓣脱垂以及主动脉返流等。目前,马凡诊断主要根据Ghent原则,只有当存在两个以上系统的体征时,才能确诊为马凡综合征,否则诊断为马凡相关疾病。但患者临床症状复杂多样且个体差异较大,因此采用该原则难以早期诊断。严重的心血管并发症是马凡综合征患者的主要致死因素,因此,尽快诊断并进行相应的预防和治疗十分重要。
马凡综合征的发生与多种基因的突变密切相关,主要是人原纤维蛋白1基因(FBN1)基因和转化生长因子β受体(TGFBR)基因,其中,以FBN1基因突变与马凡综合征相关性的研究最多。目前已有多个FBN1突变与马凡综合征相关,点突变是最常见的突变类型。现有技术CN109666729A公开了一种突变基因,在人类FBN1基础上,编码区第718位碱基C突变为T,并提供了该突变位点在制备马凡综合征筛查试剂盒中的用途。现有技术CN109554467A公开了一种突变基因,在人类FBN1基因的基础上,编码区第3617位碱基G突变为A,并提供了该突变位点在制备马凡综合征筛查试剂盒中的用途。现有技术CN110527686B则公开了一种突变基因,在人类FBN1基础上,编码区第3451位产生了杂合缺失变异,还提供了该突变位点在制备马凡综合征检测试剂盒中的用途。
马凡综合征是一种发病率低但死亡率极高的疾病,通过检测基因突变用于马凡综合征的早期诊断,从而及时进行针对性治疗,对于提高患者的生存具有重要的意义。马凡综合征作为一种罕见病,每一个基因相关突变位点的发现都极其困难,对于该疾病的基因诊断和基因治疗都具有十分重要的意义。因此,寻找新的FBN1基因的变异位点并针对该变异位点提供检测准确率、重复性高的试剂盒有助于马凡综合征的分子诊断和家系遗传分析,对其家系的优生优育和疾病防治也有重要的意义。
发明内容
本发明通过对马凡综合征家族成员的全血DNA进行碱基序列分析,在家族患病成员中检测到了FBN1基因的c.5688-5689insG碱基插入变异(p.Arg1897Glufs),家系中的未患病成员均未检测到该变异。同时对150例表型健康的对照成员和2例家族成员进行检测,也均未检测到该变异,说明该基因变异与马凡综合征的发病密切相关。
基于上述发现,本发明发现了一个新的马凡综合征相关的致病基因变异,即FBN1基因c.5688-5689(p.Arg1897Glufs)插入突变,并提供了一种基于FBN1基因c.5688-5689插入突变的马凡综合征检测试剂盒,所述的试剂盒具有较好的检测灵敏度及准确度,可用于马凡综合征患者的早期临床辅助诊断,有助于确诊患者的早期治疗,降低患病率和死亡率,对其家系的优生优育和疾病防治也有重要的意义。
为了实现上述发明目的,本发明的技术方案如下:
一方面,本发明提供了一种FBN1基因c.5688-5689(p.Arg1897Glufs)插入突变在制备马凡综合征检测试剂中的应用。
具体地,所述的c.5688-5689插入突变使FBN1蛋白第1897位的精氨酸转变为谷氨酸,其后的氨基酸移码表达,并使得在其后第31位氨基酸处提前出现一终止密码子。通过使用Alphafold软件对此蛋白质片段进行三级结构预测发现,此突变可能会导致蛋白截短表达,影响蛋白质的功能。查询Uniprot数据库发现该位点位于EGF-like 32calcium-bindingdomain,该结构域中氨基酸的改变能够破坏钙结合或二级结构的稳定性,影响钙离子的结合,从而导致FBN1蛋白质功能异常。经国际千人基因组数据库、人类全外显子组测序项目数据库ESP6500数据库和六万人外显子组整合数据库ExAC数据库检索,并查询Clinvar、HGMD数据库未发现该变异,文献检索也未发现该变异与疾病相关的报导。该位点附近的变异c.5683T>C(p.Cys1895Arg),c.5699G>A(p.Cys1900Tyr)均曾被报导为马凡综合征的致病突变。
另一方面,本发明提供了FBN1基因c.5688-5689(p.Arg1897Glufs)插入突变检测试剂在制备马凡综合征检测试剂盒中的应用。
具体地,所述的检测试剂盒为基因检测试剂盒或蛋白检测试剂盒。
具体地,所述的FBN1基因c.5688-5689(p.Arg1897Glufs)插入突变检测试剂为测序试剂或蛋白检测试剂。
进一步具体地,所述的测序试剂包括Sanger测序试剂、荧光定量PCR试剂、限制性酶切片段长度多态性方法用试剂、单链构象多态性分析用试剂和/或等位基因特异的寡聚核苷酸杂交用试剂。
又一方面,本发明提供了一种马凡综合征检测试剂盒,所述的试剂盒包括任选的用于检测FBN1基因c.5688-5689(p.Arg1897Glufs)插入突变的试剂。
具体地,所述的检测试剂盒为基因检测试剂盒或蛋白检测试剂盒。
具体地,所述的FBN1基因c.5688-5689(p.Arg1897Glufs)插入突变检测试剂为测序试剂或蛋白检测试剂。
进一步具体地,所述的测序试剂包括Sanger测序试剂、荧光定量PCR试剂、限制性酶切片段长度多态性方法用试剂、单链构象多态性分析用试剂和/或等位基因特异的寡聚核苷酸杂交用试剂。
在某些实施例中,本发明所述的用于检测FBN1基因c.5688-5689(p.Arg1897Glufs)插入突变的试剂为Sanger测序试剂,所述的Sanger测序试剂包括以下引物序列:
F(SEQ ID NO:1):TGTCCTTAGCTTGCCTCCTT;
R(SEQ ID NO:2):GGTCTCAGAATGTATCCCTCAC。
在某些实施例中,本发明还提供了一种蛋白检测试剂,所述的蛋白检测试剂包含如SEQ ID NO:8所示的氨基酸序列。
又一方面,本发明提供了一种检测FBN1基因c.5688-5689(p.Arg1897Glufs)插入突变的方法,所述的方法为非疾病诊断或治疗方法,所述的方法包括利用FBN1基因c.5688-5689(p.Arg1897Glufs)插入突变检测试剂或试剂盒对样本进行检测。
具体地,所述的方法包括以下步骤:
(1)样本DNA提取、扩增、纯化;
(2)对步骤(1)得到的纯化产物进行测序;
(3)测序结果分析。
在某些实施例中,本发明采用Sanger测序检测FBN1基因c.5688-5689(p.Arg1897Glufs)插入突变,所述的Sanger测序试剂包括以下引物序列:
F(SEQ ID NO:1):TGTCCTTAGCTTGCCTCCTT;
R(SEQ ID NO:2):GGTCTCAGAATGTATCCCTCAC。
与现有技术相比,本发明的积极和有益效果在于:
(1)本发明首次发现了FBN1基因c.5688-5689(p.Arg1897Glufs)插入突变位点可作为一种新的马凡综合征生物标志物,可以将携带FBN1基因第5688-5689位碱基变异的马凡综合征患者和正常人群区分开,对马凡综合征的临床辅助诊断和早期检测具有重要的意义,有助于确诊患者的早期治疗,降低患病率和死亡率。
(2)通过检测受试者是否携带FBN1基因c.5688-5689(p.Arg1897Glufs)插入突变,可以检测该变异的携带者,有助于马凡综合征的分子诊断和家系遗传分析,同时可以研究基因突变与临床表型之间的关系,为其将来可能需要的产前诊断提供依据,同时为马凡综合征的药物研发提供新的靶点。
(3)本发明所述的检测FBN1基因c.5688-5689(p.Arg1897Glufs)插入突变的试剂具有较好的准确性、重复性和灵敏度,操作简便,可用于大规模的生产及检测。
附图说明
图1为马凡综合征家系图。
图2为健康者的测序结果图。
图3为马凡综合征患者的测序结果图。
图4为FBN1野生蛋白1766-2012位肽段三维结构模型预测图。
图5为FBN1p.Arg1897Glufs突变蛋白1766-2012位肽段三维结构模型预测图。
图6为重组蛋白SDS-PAGE凝胶电泳分析图,其中,M:marker;1:0.5mg/mL BSA;2:FBN1野生型1766-2012位重组蛋白;3:FBN1突变蛋白(c.5688-5689insG(p.Arg1897Glufs))1766-2012位重组蛋白。
具体实施方式
下面结合具体实施例,对本发明作进一步详细的阐述,下述实施例不用于限制本发明,仅用于说明本发明。以下实施例中所使用的实验方法如无特殊说明,实施例中未注明具体条件的实验方法,通常按照常规条件,下述实施例中所使用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1.FBN1基因c.5688-5689(p.Arg1897Glufs)插入突变检测试剂盒本实施例1所述的试剂盒组分如下表1所述。
表1.试剂盒组分
实施例2.患者/携带者验证实验
1.样本采集及临床资料收集
在先证者及其家属自愿签署知情同意书的前提下,在深圳市眼科医院抽取5mL左右全血样本并于低温冷藏条件储存,建立病历资料库,详细记录先证者病情、家系情况等资料。本研究已得到深圳市眼科医院伦理委员会批准。
2.制备基因组DNA
采用市售DNA提取试剂盒对人类全血EDTA抗凝样本进行全基因组DNA的提取,对DNA的浓度和纯度进行检测。
3.PCR引物设计
根据NCBI数据库(https://www.ncbi.nlm.nih.gov/)中FBN1基因的DNA序列,在FBN1基因突变位置的上下游设计引物,具体引物序列见表2。
表2.引物信息
扩增得到的野生型序列如SEQ ID NO:3所示,突变型序列如SEQ ID NO:4所示。
4.PCR扩增试剂配置
该PCR试剂用于扩增包含目的基因位点在内的一段DNA序列,其组成见表3所示。
表3.PCR扩增试剂组成
5.目的片段扩增
将反应体系混合,在PCR仪上进行目的基因片段的扩增反应,扩增程序如下表4所示。
表4.PCR扩增程序
6.PCR产物的检测
取5μL的PCR产物,使用1.5%的琼脂糖凝胶以120V电压进行30min左右的电泳检测PCR产物,选用1000bp Marker作为参考。
7.PCR产物纯化
凝胶电泳完成后,采用市售胶回收纯化试剂盒对PCR产物进行纯化。
7.1电泳后,从琼脂糖凝胶上切下目的条带放入1.5mL离心管中。
7.2加入等体积的溶胶液(如胶为10mg,则加10μL溶胶液),50-60℃水浴加热至胶全融,期间需每隔约3min颠倒混匀,以加速凝胶融解。
7.3将溶解的凝胶混合物加入到DNA纯化柱内,室温放置1min。
7.4以16000g(约12000-14000rmp左右)离心1min,倒弃收集管内的液体。
7.5在DNA纯化柱内加入700μL洗涤液,16000g离心1min,倒弃收集管内的液体。
7.6添加500μL洗涤液,16000g离心1min,倒弃收集管内的液体。
7.7以16000g离心1min,除去残留液体并让残留的乙醇充分挥发。
7.8将DNA纯化柱置于新的1.5mL离心管上,向DNA纯化柱中央滴加20μL洗脱液放置5min。
7.9以16000g离心1min,所得液体即为高纯度DNA。
8.Sanger测序
使用AppliedBiosysterm 3500Dx系列基因分析仪进行Sanger测序。
9.数据分析
将测序数据与目的基因FBN1序列进行比对,从而验证基因变异类型。
实施例3.重复性检测
采用本发明实施例2所述的方法重复3次检测1例马凡综合征患者和1例健康者的血液样本,检测结果如下表5所示。
表5.重复性检测结果
样本 | 患者 | 健康人 |
第一次 | 阳性 | 阴性 |
第二次 | 阳性 | 阴性 |
第三次 | 阳性 | 阴性 |
上述检测结果表明,本申请所述的位点及检测引物具有较好的重复性。
实施例4.灵敏度检测
将本发明实施例2纯化的DNA产物(患者)按照梯度稀释(0.6μg、0.4μg、0.2μg),并进行检测,检测结果如下表6所示。
表6.灵敏度检测结果
样本浓度(μg) | 患者 | 健康人 |
0.6 | 阳性 | 阴性 |
0.4 | 阳性 | 阴性 |
0.2 | 阳性 | 阴性 |
上述检测结果表明,本申请所述的位点及检测引物具有较好的灵敏度。
实施例5.无关样本验证
招募1个马凡综合征家系,对所有家系成员进行体格、眼部、心血管系统检查,初步确认其符合马凡综合征的特征。通过基因检测,在该家系中检查到有1名马凡综合征病人,据家系成员介绍,该家系中还有1位已故成员也患有马凡综合征,死于突发的主动脉瘤破裂(家系图如图1所示)。
另外招募了150例未患有马凡综合征的健康人群作为对照。
使用实施例2中所述的方法扩增该家系每个成员以及对照人群的FBN1基因部分碱基片段,扩增完成后进行Sanger测序后进行分析。
其中,图2为健康者检测结果,图3为马凡综合征患者检测结果。
根据检测结果可知,马凡综合征患者FBN1基因c.5688-5689(p.Arg1897Glufs)位点发生G碱基插入突变,正常健康人样本未发现该位点突变,该结果与临床诊断结果一致。
实施例6.蛋白检测
1.通过Alphafold软件对FBN1野生和p.Arg1897Glufs突变蛋白1766-2012位肽段进行三维结构模型预测,结果显示p.Arg1897Glufs突变后蛋白质结构发生明显缩短,结果分别如图4和5所示。
2.重组蛋白制备及检测
2.1.野生型和突变型FBN1片段的原核细胞表达质粒由上海纽普生物科技有限公司根据密码子优化后的基因序列进行合成构建。其中,野生型FBN1片段的密码子优化后的基因序列如SEQ ID NO:5所示,突变型FBN1片段的密码子优化后的基因序列如SEQ ID NO:6所示。
2.2.将野生型和突变型质粒转化至大肠杆菌BL21(DE3),使用IPTG诱导野生型FBN1和突变型FBN1(c.5688-5689insG(p.Arg1897Glufs))融合蛋白的表达。
2.3.包涵体经过变复性的方式,重溶目标蛋白。具体如下:将菌体沉淀重悬于20mL裂解液,超声破碎。将超声破碎的细胞裂解液4℃10000r/min离心20min,收集沉淀。使用包涵体洗涤液洗涤包涵体3次。用溶解缓冲液溶解包涵体,4℃放置过夜;室温,10000r/min离心15min。将上述溶液滴加20mM Tris-HCl,0.15MNaCl,pH8.0缓冲液中,逐步成倍梯度稀释缓慢搅拌,将蛋白溶液装入透析袋于20mM Tris-HCl,0.15M NaCl,pH8.0溶液中透析过夜。
2.4.利用低压层析系统,对融合蛋白进行Ni柱亲和纯化。
2.5.收集纯化后的目的蛋白,进行12%SDS-PAGE凝胶电泳分析。
结果如图6所示,由图6可知,突变型的FBN1片段蛋白相比于野生型明显缩短,这表明突变后的FBN1提前终止翻译。FBN1野生型1766-2012位重组蛋白序列如SEQ ID NO:7所示,突变型(c.5688-5689insG(p.Arg1897Glufs))
1766-2012位重组蛋白序列如SEQ ID NO:8所示。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对本发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。
SEQUENCE LISTING
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gatcagacga tgtgcctgga catcaacgaa tgtgaagaac gttgcctgtg ggaatggaac 420
ctgccggaac acaactggtt tctgcagctg cctctgcagt cttggttcca cccgttctct 480
cagcag 486
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Gly Gln Cys Asn Asp Arg Asn Glu Cys Gln Glu Ile Pro Asn Ile Cys
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Ser His Gly Gln Cys Ile Asp Thr Val Gly Ser Phe Tyr Cys Leu Cys
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His Thr Gly Phe Lys Thr Asn Asp Asp Gln Thr Met Cys Leu Asp Ile
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Asn Trp Phe Leu Gln Leu Pro Leu Gln Ser Trp Phe His Pro Phe Ser
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Gln Gln
Claims (10)
1.FBN1基因c.5688-5689插入突变在制备马凡综合征检测试剂中的应用。
2.FBN1基因c.5688-5689插入突变检测试剂在制备马凡综合征检测试剂盒中的应用。
3.根据权利要求2所述的应用,其特征在于:所述的检测试剂盒为基因检测试剂盒或蛋白检测试剂盒。
4.根据权利要求3所述的应用,其特征在于:所述的FBN1基因c.5688-5689插入突变检测试剂为测序试剂或蛋白检测试剂。
5.根据权利要求4所述的应用,其特征在于:所述的测序试剂包括Sanger测序试剂、荧光定量PCR试剂、限制性酶切片段长度多态性方法用试剂、单链构象多态性分析用试剂和/或等位基因特异的寡聚核苷酸杂交用试剂。
6.一种马凡综合征检测试剂盒,其特征在于:所述的试剂盒包括任选的用于检测FBN1基因c.5688-5689插入突变的试剂。
7.根据权利要求6所述的试剂盒,其特征在于:所述的试剂盒为基因检测试剂盒或蛋白检测试剂盒。
8.根据权利要求7所述的试剂盒,其特征在于:所述的用于检测FBN1基因c.5688-5689插入突变的试剂为测序试剂或蛋白检测试剂。
9.一种检测FBN1基因c.5688-5689插入突变的方法,所述的方法为非疾病诊断或治疗方法,其特征在于:所述的方法包括利用FBN1基因c.5688-5689插入突变检测试剂或权利要求6-8所述的试剂盒对样本进行检测。
10.根据权利要求9所述的方法,其特征在于:所述的方法包括以下步骤:
(1)样本DNA提取、扩增、纯化;
(2)对步骤(1)得到的纯化产物进行测序;
(3)测序结果分析。
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CN115976187A (zh) * | 2022-10-11 | 2023-04-18 | 深圳市第二人民医院(深圳市转化医学研究院) | 一种Loeys-Dietz综合征检测试剂盒 |
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