CN108342404A - Inpp5e基因突变体及其应用 - Google Patents
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Abstract
本发明提供一种分离的编码INPP5E基因突变体的核酸,所述核酸具有以下两个位点突变:c.1524C>G和c.1688G>A。该突变体可以应用Joubert综合征的检测和治疗,具有非常广泛的临床应用价值。
Description
技术领域
本发明涉及分子生物学领域,具体涉及一种INPP5E基因突变体及其应用。
背景技术
Joubert综合征(Joubert syndrome,MIM#213300)是一种罕见的神经系统疾病,主要表现为小脑蚓部发育不全或缺如,于1969年由Joubert等首次报道,发病率估计约为1:100 000。Joubert综合征最典型的特点为小脑蚓部发育不全或缺如,临床表现主要包括阵发性呼吸过度或者呼吸暂停、共济失调、发育迟缓、眼球运动障碍、认知缺陷,部分患者常伴发视网膜缺损或视网膜发育不良、多囊肾和多指(趾)症,肝纤维囊肿等。
Joubert综合征通常由MRI结合临床表现进行确诊。1992年,Saraiva和Baraitser在总结了101个病例以后提出了Joubert综合征的诊断标准:(1)小脑蚓部发育不全;(2)肌张力低;(3)发育迟缓;(4)异常呼吸或者异常眼运动。1997年,Maria等又将影像学的“磨牙征”等加入了JS的诊断标准中。Joubert综合征主要的影像学特征性表现有:小脑蚓部部分或完全缺如、小脑上脚增粗,表现出“磨牙征”(molar tooth sign,MTS,又称“臼齿征”)“中线裂”或“蝙蝠翼”,其中“磨牙征”是Joubert综合征诊断的最重要特征。目前临床上通常把所有具有“磨牙征”的疾病统称为Joubert综合征及其相关疾病(Joubert syndromerelated disorders,JSRD)。
Joubert综合征是一种遗传性疾病。大部分为散发,其主要的遗传方式是常染色体隐性遗传,仅在少部分因OFD1基因突变导致的患者中表现为X连锁隐性遗传。目前已经报道Joubert综合征的致病基因有超过30个,分别为:INPP5E、TMEM216、AHI1、NPHP1、CEP290、TMEM67、RPGRIP1L、ARL13B、CC2D2A、OFD1、TTC21B、KIF7、TCTN1、TMEM237、CEP41、TMEM138、C5orf42、TCTN3、ZNF423、TMEM231、CSPP1、PDE6D、KIAA0586、TCTN2、CEP104、KIAA0556、B9D1、MKS1、TMEM107、ARMC9、CEP290、SUFU、PIBF1以及KIAA0753。其中,以C5orf42、CC2D2A、CEP290、AHI1以及TMEM67基因的突变最为常见。
INPP5E基因位于9号染色体,含有10个外显子,由644个氨基酸组成,该基因编码的蛋白质是磷脂酰肌醇-5-磷酸酶(INPP5E),分子量为70205Da。INPP5E的主要功能是催化PI(3.4.5)P3与PI(4,5)P2分别生成PI(3,4)P2与PI4P。INPP5E与纤毛的发生和维持机制有关。与JSRD有关的INPP5E错义突变主要集中分布在磷酸酶结构域中,影响了磷酸酶的活性,导致细胞内磷酸肌醇的浓度改变,使得纤毛在外界刺激时表现不稳定。在INPP5E突变导致的Joubert综合征患者的成纤维细胞表现出无纤毛或者短纤毛的表型,表明这些患者的纤毛发生和维持机制受到了损害。
Sanger测序是临床分子诊断的金标准,但是检测效率低、成本高,传统的酶切、杂交、TaqMan探针、DHPLC和HRM等方法针对突变热点进行检测,但检测通量较低。近几年微阵列芯片技术的发展可以同时针对数百个突变位点进行高通量检测,极大提高了诊断效率,降低了检测成本。但微阵列芯片技术存在检测周期长、检测成本相对较高,对除点突变之外的其他突变类型的检出能力有限的缺点。采用靶向测序方法(即目标区域测序)有目的地研究基因组特定区域,是一种大规模的多重PCR扩增方法,能够实现几千甚至上万重引物对进行快速扩增目标区域检测低频率或稀有突变。Joubert综合征是一种罕见的颅脑先天性发育畸形,目前发现的Joubert综合征致病基因致病位点多位于编码区,并且没有集中的突变热点。本发明采用目标区域测序的方法,对INPP5E基因在内的Joubert候选基因组合进行高通量测序,结合Sanger方法进行验证检测,确定已知突变和发现新的致病突变,确定了一种与Joubert综合征相关的基因突变体。
发明内容
在一种实施方式中,本发明提供一种分离的编码INPP5E基因突变体的核酸,所述核酸具有以下两个位点突变:c.1524C>G和c.1688G>A。
在一种实施方式中,本发明提供一种分离的多肽,所述分离的多肽由权利要求1所述的核酸编码,所述多肽具有选自下列的两种突变:p.Asp508Glu和p.Arg563His。
在一种实施方式中,本发明提供一种Joubert综合征的检测试剂盒,所述检测试剂盒包括检测INPP5E基因的以下两个位点突变的试剂:c.1524C>G和c.1688G>A。
在一种实施方式中,所述检测试剂盒是Sanger测序试剂盒、高通量测序试剂盒、基因芯片试剂盒或PCR试剂盒。
在一种实施方式中,本发明提供一种Joubert综合征治疗剂,所述治疗剂是抑制INPP5E基因的以下两个位点突变的作用剂:c.1524C>G和c.1688G>A。
在一种实施方式中,本发明提供一种筛选患Joubert综合征的生物样品的方法,所述方法包括以下步骤:从所述生物样品提取INPP5E基因核酸样本;确定所述核酸样本INPP5E基因的核酸序列;所述核酸样本INPP5E基因的核酸序列与正常人INPP5E基因相比,具有以下两个位点突变:c.1524C>G和c.1688G>A;所述两个突变都存在是所述生物样品患Joubert综合征的指示;任选地,所述生物样品为选自人体血液,任选地,所述核酸样本为全基因组DNA。
在一种实施方式中,从所述生物样品提取核酸样本进一步包括:从所述生物样品提取RNA样本,优选所述RNA样本为mRNA;以及基于所述RNA样本,通过反转录反应,获得cDNA样本,所述cDNA样本构成所述核酸样本。
在一种实施方式中,提供一种筛选患Joubert综合征的生物样品的系统,所述系统包括以下:核酸提取装置,所述核酸提取装置用于从所述生物样品提取核酸样本;核酸序列确定装置,所述核酸序列确定装置与所述核酸提取装置相连,用于对所述核酸样本进行分析,以便确定所述核酸样本的核酸序列;判断装置,所述判断装置与所述核酸序列确定装置相连,以便基于所述核酸样本的核酸序列与正常人INPP5E基因相比,是否具有以下两个位点突变:c.1524C>G和c.1688G>A突变,判断所述生物样品是否患Joubert综合征。
在一种实施方式中,本发明提供一种构建体,其包含上述分离的编码INPP5E基因突变体的核酸。
在一种实施方式中,本发明提供一种重组细胞,所述重组细胞是通过上述的构建体转化受体细胞而获得的。
附图说明
为了更清楚地说明本申请实施例中的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本申请中记载的一些实施例,对于本领域普通技术人员来说,在不付出创造性劳动的前提下,还可以根据这些附图获得其它的附图。
图1为INPP5E:NM_019892.4:c.1524C>G p.Asp508Glu的位点测序图;和
图2为INPP5E:NM_019892.4:c.1688G>A;p.Arg563His的位点测序图。
具体实施方式
为了使本领域技术领域人员更好地理解本申请中的技术方案,下面将结合下面结合实施例对本发明作进一步说明,显然,所描述的实施例仅仅是本申请一部分实施例,而不是全部的实施例。基于本申请中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其它实施例,都应当属于本申请保护的范围。下面结合附图及实施例对本发明作进一步描述。
实施例一.候选基因INPP5E测序
1.样本收集:抽取医院明确诊断为Joubert综合征的患者及其家属的外周血。外周血的采集及家系、临床资料的收集符合知情同意原则,取得受试者知情同意,血液样本编号后置-80℃低温冰箱保存。
2.外周血DNA的提取和纯化
采用QIAamp DNA Blood Mini Kit试剂盒(Qiagen,德国)的操作说明从全血中提取基因组DNA,利用Qubit2.0(Invitrogen,美国)进行定量。
a)向1.5ml离心管加入20ul蛋白酶K
b)加入200ul buffy coat(或全血样本)。如果体积不足200ul,用PBS补足。
c)加入200ul buffer AL,漩涡振荡15sec。
d)56℃水浴10min
e)快速离心,避免管盖上留有溶液。
f)加入200ul无水乙醇,漩涡振荡15sec,快速离心。
g)将所有溶液转移到QIAamp离心柱中,10000rpm离心2min,将离心柱转入新的收集管。
h)向离心柱上加入500ul buffer AW1,10000rpm离心2min,将离心柱转入新的收集管。
i)向离心柱上加入500ul buffer AW2,10000rpm离心5min,弃收集管。将离心柱转入新的收集管,10000rpm离心3min。
j)将离心柱放入新的1.5ml离心管,向离心柱上加入200ul buffer AE,室温放置5min,10000rpm离心2min。
k)弃去离心柱,盖上管盖。保存于-20度冰箱。
l)DNA定量:通常能得到20-50ng/μLDNA,纯度(紫外260OD:280OD)在1.8-2.0范围内。
3.测序
(1)文库构建
采用Ion AmpliSeqTM自定义Panel,对人的INPP5E基因进行高效富集,然后在IonTorrent PGM平台上进行高通量、高深度测序。建库和捕获实验采用Ion AmpliSeqTMLibraryKit建库试剂盒,严格使用说明书推荐的试剂和耗材,并参照最新的经过优化的实验流程进行操作。
实验基本流程:将基因组DNA采用磁珠法(AMPure Beads,Invitrogen,美国)纯化后,采用Ion AmpliSeq Panel扩增目的基因,在片段两端分别连接Barcode和测序引物,经PCR线性扩增后进行文库质检,合格即可进行上机测序。
(2)文库质检
文库构建完成后,使用Agilent 2100对文库的长度进行检测,符合预期后,稀释文库至15ng/mL。
(3)上机测序
库检合格,根据文库的有效浓度及数据产出需求进行Ion Torrent PGM平台测序。
4.数据分析与处理
测序产出的大量数据利用Ion ReporterTM软件经初步质量控制后,进行数据评估以确保数据的可信度,主要包括数据量、重复率、捕获效率、测序深度及覆盖率等指标,继而以GRCh37.1/hg19基因组为参考序列进行比对,对变异检测和注释,主要针对与人类基因组对比后获得的各类变异,主要包括SNP和InDel。针对性地分析主要突变对蛋白结构及功能影响的注释,常用到的软件包括SIFT、PolyPhen2和MutationTaster。
5.分析结果
经过数据筛选、深度加工和生物信息学序列比对,最终分析发现患病者INPP5E基因具有两个突变位点,NM_019892.4:c.1524C>G;p.Asp508Glu和NM_019892.4:c.1688G>A;p.Arg563His,均为错义突变。具体情况见表1。
表1.INPP5E基因突变检测结果
实施例二.Sanger测序进行突变位点的验证
1.采集实施例1中家系中3个样本的血样,提取DNA样本同实施例1;
2.PCR扩增
PCR引物设计:引物由华大六合科技有限公司合成,共设计2对引物,如表2所示。
表2.引物序列
PCR反应体系如表3所示,25μL体系的配制:
表3.反应体系
PCR反应退火温度为58℃。
3.测序:扩增反应产物经琼脂糖凝胶电泳检测后由ABI 3730xl完成Sanger测序。
4.测序结果:使用Chromas软件查看Sanger测序峰图,用SeqMan软件比对Sanger测序结果。Sanger测序结果表明,患者的INPP5E基因上同时携带这两个突变的杂合突变,父母只是其中一个杂合突变的携带者,患者的c.1524C>G位点来自于母亲,c.1688G>A位点来自于父亲。两个杂合突变同时发生时致病。Sanger测序结果与候选基因测序结果相符合。具体结果图1与图2。
实施例三.基因突变检测试剂盒的制作
突变位点试剂盒的制作和操作流程基于Sanger测序技术。试剂盒含有实施例二中扩增INPP5E基因两个突变位点的特异性引物,该试剂盒还可以包括PCR反应常用的试剂,如Taq酶、dNTP混合液、MgCl2溶液、去离子水等;这些常用试剂都是本领域技术人员熟知的,另外还可以含有标准品和/或对照品(如确定基因型的标准品和空白对照等)。此试剂盒的价值在于只需要外周血而不需要其它组织样品,通过最精简和特异的引物对检测突变位点,再通过突变位点谱辅助判断Joubert综合征,不仅稳定,检测方便,且精确,大大提高疾病诊断的敏感性和特异性,因此将此试剂盒投入实践,可以帮助指导诊断和更有效的个体化治疗。
应该理解到披露的本发明不仅仅限于描述的特定的方法、方案和物质,因为这些均可变化。还应理解这里所用的术语仅仅是为了描述特定的实施方式方案的目的,而不是意欲限制本发明的范围,本发明的范围仅受限于所附的权利要求。
本领域的技术人员还将认识到,或者能够确认使用不超过常规实验,在本文中所述的本发明的具体的实施方案的许多等价物。这些等价物也包含在所附的权利要求中。
Claims (10)
1.一种分离的编码INPP5E基因突变体的核酸,其特征在于,所述核酸具有以下两个位点突变:c.1524C>G和c.1688G>A。
2.一种分离的多肽,其特征在于,所述分离的多肽由权利要求1所述的核酸编码,所述多肽具有选自下列的两种突变:p.Asp508Glu和p.Arg563His。
3.一种Joubert综合征的检测试剂盒,其特征在于,所述检测试剂盒包括检测INPP5E基因的以下两个位点突变的试剂:c.1524C>G和c.1688G>A。
4.根据权利要求3所述的检测试剂盒,其特征在于,所述检测试剂盒是Sanger测序试剂盒、高通量测序试剂盒、基因芯片试剂盒或PCR试剂盒。
5.一种Joubert综合征治疗剂,其特征在于,所述治疗剂是抑制INPP5E基因的以下两个位点突变的作用剂:c.1524C>G和c.1688G>A。
6.一种筛选患Joubert综合征的生物样品的方法,其特征在于,包括以下步骤:
从所述生物样品提取INPP5E基因核酸样本;
确定所述核酸样本INPP5E基因的核酸序列;
所述核酸样本INPP5E基因的核酸序列与正常人INPP5E基因相比,具有以下两个位点突变:c.1524C>G和c.1688G>A;所述两个突变都存在是所述生物样品患Joubert综合征的指示,
任选地,所述生物样品为选自人体血液,
任选地,所述核酸样本为全基因组DNA。
7.根据权利要求6所述的方法,其特征在于,从所述生物样品提取核酸样本进一步包括:从所述生物样品提取RNA样本,优选所述RNA样本为mRNA;以及基于所述RNA样本,通过反转录反应,获得cDNA样本,所述cDNA样本构成所述核酸样本。
8.一种筛选患Joubert综合征的生物样品的系统,其特征在于,包括:
核酸提取装置,所述核酸提取装置用于从所述生物样品提取核酸样本;
核酸序列确定装置,所述核酸序列确定装置与所述核酸提取装置相连,用于对所述核酸样本进行分析,以便确定所述核酸样本的核酸序列;
判断装置,所述判断装置与所述核酸序列确定装置相连,以便基于所述核酸样本的核酸序列与正常人INPP5E基因相比,是否具有以下两个位点突变:c.1524C>G和c.1688G>A突变,判断所述生物样品是否患Joubert综合征。
9.一种构建体,其特征在于,包含权利要求1所述的分离的编码INPP5E基因突变体的核酸。
10.一种重组细胞,其特征在于,所述重组细胞是通过权利要求9所述的构建体转化受体细胞而获得的。
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