CN117947153A - 线粒体病相关iars2基因突变位点及其应用 - Google Patents
线粒体病相关iars2基因突变位点及其应用 Download PDFInfo
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Abstract
本发明公开了线粒体病相关IARS2基因突变位点及其应用,属于生物技术领域,该突变位点包括c.1_390del、c.2090G>A、c.2122G>A和c.2450G>A中的一种或多种。本发明发现了IARS2基因缺陷导致线粒体病发生的分子机制,并发现了IARS2缺陷相关线粒体病的4个新变异位点(c.1_390del、c.2090G>A、c.2122G>A和c.2450G>A),通过功能分析验证了位点的致病性,拓展了IARS2致病基因突变谱,为IARS2缺陷相关线粒体病的临床诊断和预测提供重要参考。
Description
技术领域
本发明涉及生物技术领域,特别是涉及线粒体病相关IARS2基因突变位点及其应用。
背景技术
Leigh综合征(LS),又称亚急性坏死性脑病,是由线粒体或核基因突变引起的儿童期最常见的线粒体疾病。LS由Denis Archibald Leigh于1951年首次报道,患病率约为1:40,000。虽然LS的发病率低,但临床症状迅速恶化,并可能导致婴儿期死亡。LS主要特点是中枢神经系统(包括脑、脊髓和视神经)退行性变,表现为运动技能丧失、食欲不振、呕吐、易怒和/或癫痫发作,以及全身无力、肌张力缺乏、乳酸性酸中毒,进而导致心、肝、胃肠和肾小管功能障碍。目前已经报道了超过75个基因缺陷导致LS发生。LS与氧化磷酸化系统(OXPHOS)密切相关,80%的LS患者存在呼吸链酶复合物缺乏。虽然五种OXPHOS复合物缺陷都能导致LS发生,但复合物I缺乏最为常见,单独的复杂IV缺陷和多种OXPHOS缺陷也较常见,这为LS的临床诊断提供了生化缺陷的证据。
IARS2编码线粒体异亮氨酰-tRNA合成酶,其是I类线粒体氨酰-tRNA合成酶(ARS)。ARS家族有37个成员,与细胞质或线粒体翻译相关。编码ARS的基因突变可产生高度多样的临床表型,影响代谢需求特别高的组织。作为核编码的线粒体蛋白,IARS2需要从细胞质输入到线粒体中,并催化异亮氨酸残基与同源mt-tRNA的连接。据报道,IARS2缺乏可导致白内障、生长激素缺乏、感觉神经病、感觉神经性听力损失和LS,但IARS2突变的发病机制和分子机制尚不清楚,还需进一步的探索研究。迄今为止,全球已报告28例IARS2突变病例,发现25种变异,其中14种表现出LS特征。然而,IARS2缺陷导致线粒体病发生的分子机制尚不清楚。
发明内容
本发明的目的是提供线粒体病相关IARS2基因突变位点及其应用,以解决上述现有技术存在的问题,本发明发现了IARS2基因缺陷导致线粒体病发生的分子机制,并发现了IARS2缺陷相关线粒体病的4个新变异位点,通过功能分析验证了位点的致病性,拓展了IARS2基因突变谱,为IARS2缺陷相关线粒体病的临床诊断和预测提供重要参考。
为实现上述目的,本发明提供了如下方案:
本发明提供线粒体病相关IARS2基因突变位点,所述IARS2基因突变位点包括c.1_390del、c.2090G>A、c.2122G>A和c.2450G>A中的一种或多种;
所述c.2090G>A、c.2122G>A和c.2450G>A突变位点依次位于IARS2参考转录组序列的第2090、2122和2450位碱基处;其中,所述c.2090G>A、c.2122G>A和c.2450G>A突变位点碱基均突变为A;
所述c.1_390del突变位点的缺失片段位于IARS2参考转录组序列的第1-390位碱基处;
所述IARS2参考转录组序列登录号为NM_018060.4。
本发明还提供一种用于检测上述IARS2基因突变位点的试剂在制备IARS2基因缺陷相关线粒体病辅助诊断或预测试剂盒中的应用。
进一步地,所述试剂包含引物组。
进一步地,所述引物组包括:
检测所述c.2090G>A、c.2122G>A和c.2450G>A突变位点的如SEQ ID NO:1-2所示的引物;检测所述c.1_390del突变位点的如SEQ ID NO:3-4所示的引物。
进一步地,若所述c.2090G>A、c.2122G>A和c.2450G>A突变位点均突变为A,和/或所述c.1_390del突变位点缺失目的片段,判断患者罹患IARS2基因缺陷相关线粒体病;
所述目的片段为IARS2参考转录组序列的第1-390位碱基片段;所述IARS2参考转录组序列登录号为NM_018060.4。
本发明还提供一种辅助诊断和预测IARS2基因缺陷相关线粒体病的试剂盒,包含上述的引物组。
本发明公开了以下技术效果:
本发明发现了IARS2基因缺陷导致线粒体病发生的分子机制,并发现了IARS2缺陷相关线粒体病的4个新变异位点(c.1_390del、c.2090G>A、c.2122G>A和c.2450G>A),通过功能分析验证了位点的致病性,拓展了IARS2基因突变谱,为IARS2缺陷相关线粒体病的临床诊断和预测提供重要参考。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,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发明的一些实施例,对于本领域普通技术人员来讲,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他的附图。
图1为患者1和患者2的家系图谱(A)和一代测序结果(B);A中黑色箭头表示先证者,方框表示男性,圆圈表示女性,黑色实心方框表示男性患者;
图2为患者2在IARS2外显子1和2中存在杂合突变的定量PCR结果;
图3为c.2450G>A(p.R817H)、c.2122G>A(p.E708K)和c.2090G>A(p.R697S)在不同物种间氨基酸的保守性(A)及等位基因频率分析结果(B);
图4为患者来源永生化B淋巴细胞的IARS2蛋白(A)及OXPHOS复合体(B)含量及定量分析结果;
图5为患者来源永生化淋巴细胞的基础氧呼吸和ATP依赖的氧呼吸(A)及细胞内ATP水平(B)和线粒体膜电位MMP含量(C)的分析结果;
图6为HEK293T IARS2敲低细胞模型的IARS2蛋白表达水平(A)以及OXPHOS复合体含量分析结果(B);
图7为HEK293T IARS2敲低细胞模型的基础氧呼吸和ATP依赖的氧呼吸(A)及细胞内ATP水平(B)和MMP含量(C)分析结果;
图8为在HEK293TIARS2敲低细胞中再表达野生型IARS2和携带c.1_390del、c.2090G>A、c.2122G>A和c.2450G>A变异的IARS2蛋白表达(A,C)及定量分析(B,D)结果;
图9和图10为在HEK293T IARS2敲低细胞中再表达野生型IARS2和携带c.2090G>A、c.2122G>A和c.2450G>A变异的线粒体功能分析结果;图9中A-B:OXPHOS复合物含量分析;图10中A:基础氧呼吸和ATP依赖的氧呼吸;B:细胞ATP含量。
具体实施方式
现详细说明本发明的多种示例性实施方式,该详细说明不应认为是对本发明的限制,而应理解为是对本发明的某些方面、特性和实施方案的更详细的描述。
应理解本发明中所述的术语仅仅是为描述特别的实施方式,并非用于限制本发明。另外,对于本发明中的数值范围,应理解为还具体公开了该范围的上限和下限之间的每个中间值。在任何陈述值或陈述范围内的中间值,以及任何其他陈述值或在所述范围内的中间值之间的每个较小的范围也包括在本发明内。这些较小范围的上限和下限可独立地包括或排除在范围内。
除非另有说明,否则本文使用的所有技术和科学术语具有本发明所述领域的常规技术人员通常理解的相同含义。虽然本发明仅描述了优选的方法和材料,但是在本发明的实施或测试中也可以使用与本文所述相似或等同的任何方法和材料。本说明书中提到的所有文献通过引用并入,用以公开和描述与所述文献相关的方法和/或材料。在与任何并入的文献冲突时,以本说明书的内容为准。
在不背离本发明的范围或精神的情况下,可对本发明说明书的具体实施方式做多种改进和变化,这对本领域技术人员而言是显而易见的。由本发明的说明书得到的其他实施方式对技术人员而言是显而易见得的。本发明说明书和实施例仅是示例性的。
关于本文中所使用的“包含”、“包括”、“具有”、“含有”等等,均为开放性的用语,即意指包含但不限于。
实施例1
材料与方法
一、材料
HEK293T细胞:购买自上海中科院,并通过STR细胞鉴定;患儿及年龄相当的健康人来源的淋巴细胞:由本实验室自行构建,所有纳入研究的人员已获得本人或其法定监护人的知情同意,并通过北京大学附属第一医院的委员会批准(伦理审批书:2017-217);B95-8细胞:购买自上海中科院,并通过STR细胞鉴定;pLVX-hyg质粒、plko.1-puro质粒、pMD2.G质粒和psPAX2质粒:购买自擎科生物技术有限公司,并通过质粒测序和序列比对;DH5α菌株和DMT菌株:由温州医科大学检验医学院和生命科学院谭国强研究员实验室惠赠。
二、方法
1病例收集
收集2020年4月-2022年1月就诊于北京大学附属第一医院儿科临床疑似“线粒体病”的443例儿童患者(<18岁)的静脉全血样本,提取DNA进行二代测序,根据致病基因筛选标准筛选候选致病基因为IARS2的患者及家系成员纳入研究。收集1例与纳入患者年龄相当的健康人纳入研究作为对照。本研究的伦理经北京大学附属第一医院伦理委员会同意(伦理审批书:2017-217)。线粒体病的诊断参考线粒体医学协会的共识声明,候选致病性变异筛选标准参考美国医学遗传学与基因组学学会指南。
2全外显子测序及线粒体全基因组测序
本研究所有样本的全基因组测序、全外显子测序及线粒体全基因组测序由北京希望组生物科技有限公司和北京优乐复生公司完成。
2.1实验步骤
2.1.1全基因组和全外显子测序
(1)DNA样本检测:按照标准流程提取全血样本中的DNA,利用Nanodrop 2000核酸蛋白分析仪进行DNA纯度检测(A260/A280比值);利用琼脂糖凝胶电泳进行DNA片段大小检测,质控其降解程度;利用Qubit荧光定量进行DNA浓度检测;
(2)文库构建:DNA质控合格后会对其进行打断、末端修复、加“A”、加Gene+Seq测序仪特有接头,并且通过PR富集构建DNA文库。然后DNA文库与探针进行液相杂交,探针为标记有生物素的DNA单链探针。最后将双链的靶区域文库进行变性、环化和消化,得到单链环状DNA。单链环状DNA通过滚环扩增技术,得到DNA纳米球。文库构建完成后,利用Qubit进行定量质控,文库质控合格后进行上机测序。
(3)上机测序:将制备好的DNA加载到微阵列芯片上,利用联合探针锚定聚合技术进行测序,将测序引物锚定分子和荧光探针在DNA纳米球进行聚合反应之后,利用高分辨成像系统对光信号进行采集、读取和识别,获得单个碱基序列信息,下个循环获得下一个碱基序列信息。
(4)生物信息学分析:包括原始下机数据过滤、数据比对和统计、成对样本变异检测和注释、突变频谱和变异特征分析、显著突变基因分析、驱动基因筛选和拷贝数变异分析等。
2.1.2线粒体全基因组测序
(1)实验流程按照Illumina公司的标准protocol执行,包括样本质量检测、文库构建、文库质量检测和文库测序等流程。具体操作:使用CTAB法提取的基因组DNA经检测合格后,用机械打断的方法使DNA片段化,然后对片段化的DNA进行片段纯化、末端修复、3’端加A和连接测序接头,再用琼脂糖凝胶电泳进行片段大小选择,进行PCR扩增形成测序文库,建好的文库进行文库质检,质检合格后用IlluminaNovaSeq进行测序。
(2)生物信息学分析流程包括:筛选有效测序数据、SPAdes组装、比对近缘物种线粒体基因组、确定目标序列、连接关系确定、形成完整的线粒体基因组序列和基因组注释。
3IARS2基因变异位点生物信息学分析
对所有变异位点的等位基因频率、变异致病性及变异后蛋白质稳定性进行数据库预测,所有数据库和网站包括:gnomAD(https://gnomad.broadinstitute.org/)、Polyphen-2(http://genetics.bwh.harvard.edu/pph2)、MuPro(http://mupro.proteomics.ics.uci.edu/)和Auto-Mute(http://binf.gmu.edu/automute/)。
4永生化B淋巴细胞系的构建
(1)将4mL肝素抗凝的外周静脉血与RPMI-1640培养基按照1:1比例稀释;
(2)缓慢轻柔地将稀释后的静脉全血加到4mL淋巴细胞分离液上(避免冲破分离界面),高速冷冻离心机(Heraeus Multifuge X1R)2500×rpm,20min,室温离心,离心机升降速1rpm/min;
(3)取血浆与淋巴细胞分离液中间的白膜层于新15mL离心管中,PBS缓冲液定容到10mL重悬白膜层,1500×rpm,10min,室温离心,离心机升降速5rpm/min;
(4)弃上清液后,用10mLPBS缓冲液重悬细胞颗粒,1000×rpm,5min,室温离心,离心机升降速5rpm/min;
(5)弃PBS缓冲液,用2mL RPMI-1640完全培养基重悬单个核细胞后加到T25瓶中,同时从-80℃取出2mL冻存的EB病毒液,37℃水浴锅中快速融化后加到细胞中,加入2μL环孢霉素A和20μL植物血凝素A,37℃,5%CO2培养箱中培养;
(6)第2周开始用20%FBS RPMI-1640完全培养基补液,隔天补一次,每次2mL,补3次;
(7)补液完成后用10%FBS RPMI-1640完全培养基培养,细胞密度足够时半定量换液或换代培养。
5DNA提取及一代测序
利用基因组DNA小量提取试剂盒法提取培养细胞中总DNA及外周静脉全血中总DNA。
将提取的DNA样本送擎科生物科技有限公司测序,测序所用引物见表1。
表1一代测序引物序列
6IARS2敲低细胞模型(IARS2-KD)的构建
6.1shRNA设计
a.将目的基因序列输入shRNA网站(https://rnaidesigner.thermofisher.com)设计shRNA,综合分析选择得分最高的shRNA序列;
b.杭州擎科基因公司合成shRNA序列,本课题使用的shRNA序列如表2。
表2 IARS2-KD shRNA序列
6.2shRNA退火
a.ddH2O分别溶解两组shRNA单链,溶解终浓度10μg/μL;
b.配制退火反应体系:正向shRNA单链10μL,反向shRNA单链10μL;
c.用微量移液枪混匀反应体系,按照表3反应条件退火;
表3 IARS2-KD shRNA退火反应条件
d.退火产物用ddH2O稀释100倍备用或-20℃保存。
6.3载体质粒线性化
选择慢病毒载体plko.1-puro作为质粒载体,本发明采用AgeI和EcoRI内切酶双酶切plko.1-puro载体,将退火后的双链shRNA连接至U6逆转录启动子。之后利用二代慢病毒包装骨架质粒psPAX2和病毒包膜表达质粒pMD2.G,共同辅助目的质粒整合进宿主基因产生病毒。
6.4目的载体构建
a.将退火后的双链shRNA按照1:100比例稀释,浓度为100ng/μL;
b.按照线性化载体:shRNA双链=1:3(pmol)进行连接。
6.5目的载体转化、筛选及鉴定
取5μL连接产物转入到50μLDH5α感受态细胞中,筛选阳性克隆。
6.6IARS2敲低稳转细胞株构建及鉴定
按照质粒小量提取试剂盒说明书提取质粒,并通过细胞转染收集病毒液:
a.扩培pMD2.G和psPAX2产病毒辅助质粒,提取质粒DNA并测定质粒浓度,4℃备用;
b.提前48h将HEK293T细胞铺在6孔细胞培养板中,使其密度50%左右,转染前将完全培养基换成基础培养基;
c.使用基础DMEM 125μL分别稀释lip3000和P3000(3.75μL、5μL);
d.按照目的质粒:pMD2.G:psPAX2=4:3:1,将总量2.5μg的质粒加入到稀释后的P3000中;
e.在已稀释的Lip3000试剂中加入稀释的DNA,用微量移液枪混匀,室温孵育10-15分钟;
f.加入DNA-脂质复合物到293T细胞中,6h之后加血清240ul;
g.收集转染后48h病毒上清液,4℃暂存,补充完全培养基后,再次收集24h病毒液上清,于48h上清液混合,0.45μm过滤器过滤后4℃备用;
6.7细胞感染:
a.提前48h将293T细胞铺在6孔细胞培养板中,使其密度70%左右时进行病毒感染;
b.按照病毒液上清:基础DMEM培养基=1:1更换细胞培养基,使其充分感染细胞;
c.6h之后加血清240ul,培养箱培养。
6.8Puro抗性筛选获得IARS2敲低细胞模型鉴定模型:
a.持续用2ug/ml Puro药杀细胞,6天后收集细胞沉淀;
b.用Western blot鉴定IARS2敲低细胞株,胶片曝光蛋白条带与对照相比降低的细胞株为成功敲低细胞株;
将成功的敲低细胞株扩大培养,并冻存于液氮中保种。
7IARS2定点突变细胞模型的构建及鉴定
提取细胞总RNA并经逆转录获得IARS2 cDNA,在目的基因cDNA两端设计反向互补寡核苷酸,并在寡核苷酸两端设计16-21bp与载体质粒完全互补的序列,擎科生物技术公司合成引物,本实验所用引物为:F:5'-atttccggtgaattcctcgagATGCGTTGGGGGCTGCGC-3',R:5'-ggagggagaggggcgggatccCTATTTTCCACTGACAACTTCTGCA-3';
按照2×Flash预混液说明书进行PCR扩增,扩增体系为:ddH2O 9.5μL、2×Flash预混液12.5μL、上、下游引物(10μM)各1μL、cDNA 1μL(不超过PCR反应总体积的1/10)。扩增程序为:98℃30s;98℃10s,65℃5s,72℃5s,共34个循环;72℃1min。
采用1%琼脂糖凝胶电泳分离鉴定PCR产物;将样本吸取一定量送擎科基因合成科技有限公司测序,将序列结果与IARS2参考序列比对(NM_018060.4),确定所有碱基与参考序列匹配。
采用PLVX-hyg载体作为目的载体,将目的基因全长cDNA连接到CMV启动子以后,从而起到过表达目的基因的目的。方法如下:
按照XhoI内切酶说明书酶切pLVX-hyg质粒,再按照BamhI内切酶说明书进行双酶切,1%琼脂糖凝胶电泳鉴定酶切效率,按照胶回收试剂盒回收正确位置的酶切载体;按照ClonExpress II一步法克隆试剂盒说明书进行酶切载体与目的cDNA连接,鉴定正确的质粒,并进行产物转化、目的菌扩增及鉴定,收集阳性菌液(PLVX-hyg-IARS2菌液)。
定点突变引物设计与目的质粒扩增:
根据突变位点位置设计引物,突变后的位点设计在上下游引物之中,设计方式为:5’-15-21bp方向互补区域+至少15bp的非互补区域-3’,现象互补区域GC含量40%-60%最佳,避免重复序列,待突变位点至引物3’端Tm值>60℃最佳。本研究所用引物由擎科基因合成公司合成,所有定点突变引物序列见表4;
表4定点突变引物序列
摇匀MutII快速突变试剂盒II中各组分,按照表5配制反应体系:
表5目的质粒扩增反应体系
微量移液器混匀反应体系,按照表6条件扩增目的质粒:
表6目的质粒扩增条件
取少量扩增产物进行琼脂糖凝胶电泳,如目的质粒条带清晰,进行质粒转化;
c.1_390del突变则利用引物F:ATTTCCGGTGAATTCCTCCTCGAGATTTTGAAAGACATAGCCAATCGA和R:GGAGGGAGAGGGGCGGGATCCCTATTTTCCACTGACAACTTCTGCA,通过PCR扩增获得,PCR反应体系和扩增条件如上。
制备DMT感受态作为突变质粒转化菌株,转化后进行目的菌培养及鉴定,筛选阳性菌液;
将扩增质粒的一代测序结果与IARS2参考序列进行比对,确定存在定点突变。将携带定点突变的质粒和IARS2过表达质粒转染到HEK293TIARS2-KD细胞模型。之后利用嘌呤霉素和潮霉素筛选携带定点突变质粒和IARS2过表达质粒的细胞模型,利用SDS-PAGE在蛋白质水平对定点突变细胞株进行基因表达验证。
8SDS-PAGE蛋白质印迹
a.收集细胞并用预冷的PBS洗涤。使用补充有ImM PMSF(Sigma-Aldrich)的RIPA裂解缓冲液(Cell signaling technology,USA)提取蛋白质。
b.将混合物在冰上孵育15min,并在4℃下以14,000×g离心10min。使用PierceBCA蛋白测定试剂盒(Thermo Fisher Scientific)测量样品的浓度。
c.将蛋白质裂解物用5×上样缓冲液在95℃下变性5min,并上样到10%蛋白质凝胶上。将蛋白质转移到0.22μmPVDF膜(BIO-RAD,USA)上,并用5%脱脂牛奶封闭1h。
d.一抗/二抗孵育。使用Super-Signal West化学发光底物(BIO-RAD)使蛋白质信号可视化,并在Gel-Pro分析仪4.0(USA)中定量灰度测量。抗体如下:IARS2(Proteintech,USA,1:2000),β-actin(Abcam,UK,1:2000),Anti-mouse IgG,HRP(Cell Signalingtechnology,1:2000)andAnti-rabbit IgG,HRP(Cell Signaling technology,1:2000).
9.蓝色非变性聚丙烯酰胺凝胶电泳(BN-PAGE)
a.使用20%Triton-100(Sigma-Aldrich)裂解细胞20分钟,并在4℃下以20,000×g离心20分钟。
b.使用Pierce BCA蛋白测定试剂盒(Thermo Fisher Scientific)测量样品的浓度,并加入6×loading以制备样品。
c.使用蓝色非变性PAGE(3.5%-16%)分离样品,并印迹到0.22μm PVDF(BIO-RAD,USA)上。然后与一抗二抗孵育。使用抗体为anti-GRIM19(Abcam,UK,1:1000),anti-SDHA(Abcam,UK,1:3000),anti-UQCRC2(Abcam.UK,1:2000),anti-MT-COI(Abcam,UK,1:2000),and anti-ATP5A(Abcam,UK,1:3000)for complex I-V。
10.氧消耗率(OCR)
使用Oxygraph-2k(Oroboros,Innsbruck,Austria)测量细胞的氧消耗。将约1×107个细胞接种于细胞培养皿中,当细胞密度达到约80%时,收集细胞并用PBS洗涤。将细胞轻轻且快速地添加到机器中。记录基础呼吸后,加入2μg/mL寡霉素(Sigma-Aldrich)以测量细胞的ATP相关线粒体呼吸。
11.细胞ATP检测
通过使用ATP生物发光测定试剂盒(Sigma-Aldrich)测量细胞ATP。收集5×106个细胞,并用预冷的磷酸盐缓冲盐水溶液(PBS)洗涤。然后用ATP提取溶液(100mM Tris-base,4mM EDTA-Na2,pH7.75)重悬细胞。将细胞煮沸90s,然后以10,000×g离心60s并收集上清液。最后,将上清液与荧光素酶测定缓冲液混合,检测自发荧光。用蛋白质浓度校正结果。用BCA蛋白浓度测定试剂盒测量样品的蛋白浓度作为校准。
12.线粒体ROS检测
通过使用MitoSOXTM Red试剂(Thermo Fisher Scientific)测量线粒体ROS含量。将约3×106个细胞接种在六孔板中,细胞密度达到约80%后,用PBS洗涤细胞,并用含有5uMMitoSOX试剂的工作液更换培养基。然后在37℃下在黑暗中孵育10分钟,轻轻收集细胞并用PBS洗涤3次。在510nm处检测激发荧光,在580nm处检测发射荧光。对于永生化淋巴细胞,收集约3×106个细胞,用含5μM MitoSOX试剂的工作液重悬,37℃避光孵育10min,轻轻收集细胞,用PBS洗涤3次,检测荧光。
13.线粒体膜电位(MMP)检测
通过使用四甲基罗丹明(Thermo Fisher Scientific)测量MMP。将约3×106个细胞接种在六孔板中,当细胞密度达到约80%时,我们用PBS洗涤细胞,并将培养基更换为含有30nM四甲基罗丹明的DMEM,然后在37℃下避光孵育30min,轻轻收集细胞并用PBS洗涤3次。在488nm处检测激发荧光,在570nm处检测发射荧光。对于永生化淋巴细胞,收集了约3×106个细胞,用含30nM四甲基罗丹明的DMEM重悬,在37℃黑暗条件下孵育30min,轻轻收集细胞,用PBS洗涤3次,然后检测荧光。
14.统计方法
所有实验均至少进行三次独立重复,利用GraphPad Prism 8对所有实验结果进行统计学分析,统计分析结果表现为均数±标准差(mean±SD),统计学方法包括独立重复样本t检验或方差分析(ANOVA)。P<0.05表示具有统计学意义,*P<0.05,**P<0.01,***P<0.001。
三、实验结果
1临床病例收集及基因型和临床表型分析
按照美国医学遗传学和基因组学学院(American college of medicalgenetics,ACMG)线粒体病诊断的专家共识中的临床诊断标准,本发明收集了2020年4月-2022年1月就诊于北京大学附属第一医院儿科的443名临床诊断或疑似线粒体病的患者的全血样本,提取血样进行二代测序和线粒体全基因组测序,根据ACMG序列变异解释的标准与指南筛选出携带IARS2基因突变的患者2例。2个病例中共发现4个变异位点,分别是c.1_390del、c.2090G>A、c.2122G>A和c.2450G>A。
患者1是一名5岁男孩,出生在一个没有遗传代谢疾病家族史的中国家庭,体重20公斤,头围52厘米,身高113厘米(图1中A)。他是G1P1,足月正常分娩,出生时体重4000克。患者在一岁之前经常感冒,并在一岁半时被送入医院。患者2至3岁时脚部容易出现脱皮症状,维生素B治疗后症状得到缓解。3岁10个月时,患者因高烧再次入院,出现虚弱和站立困难,脑部核磁共振诊断为脑炎。4岁半时,患者出现上肢、头部和面部间歇性抽搐,脑电图显示脑波异常。脑部核磁共振成像显示侧脑室尾核和纹状体长T1和T2信号增强,双侧基底节萎缩和大脑沟回扩大。实验室检查显示血氨正常(32μm,正常范围18-60μm)。随后进行了全外显子组测序以检测致病基因。在符合既定标准的筛选后,发现IARS2基因中两个新的位点变异(c.2090G>A和c.2122G>A),未检测到临床意义的线粒体基因组相关变异。分离分析证实c.2090G>A突变来自患者母亲,c.2122G>A突变来自患者父亲(图1中B)。
患者2是一名4岁男孩,出生在一个健康的中国家庭(图1中A)。他是G3P3,足月正常分娩,出生时体重4350克。患者在一岁半时被送入医院,体重13公斤,头围46厘米,身高73厘米,无法说话,很难稳坐,无法独立行走或进食,被诊断为生长迟缓。在3岁时再次入院,被诊断为症状性运动障碍和神经发育迟缓。实验室检查显示血乳酸增高(6.24mM,正常范围0.50-2.20mM),β-羟基丁酸(1.05mM,正常范围0.02-0.27mM)和肌酸激酶(1854.59U/L,正常范围50-319U/L)升高。脑部核磁共振成像显示侧脑室尾核和纹状体长T1和T2信号增强,大脑白质减少,双侧基底节对称性损伤和大脑发育异常,被诊断为Leigh综合征。随后进行了全基因组测序以检测致病基因。在符合既定标准的筛选后,发现IARS2基因中一个新的位点变异(c.2450G>A)和chr1:g.220267444_220269568的缺失,该缺失对应于IARS2外显子1和外显子2完全缺失,位于IARS2参考转录组序列的第1-390位碱基处(c.1_390del)。未检测到临床意义的线粒体基因组相关变异。分离分析证实了c.2450G>A来自患者母亲,c.2450G>A来自患者父亲(图1中B)。如图2所示,CNV-Seq发现患者在IARS2外显子1和2中存在杂合突变。定量PCR结果显示,与同龄对照组和患者母亲相比,患者及其父亲的IARS2外显子1和2的mRNA水平显著降低。
2变异位点等位基因频率及变异致病性预测
进行了一系列生物信息学分析以研究上述IARS2变体(c.2450G>A,c.2122G>A和c.2090G>A)的致病性。首先,分析了不同物种间氨基酸的保守性,结果表明c.2450G>A(p.R817H)、c.2122G>A(p.E708K)和c.2090G>A(p.R697S)在不同物种间具有高度保守性(图3中A)。此外,对这些变体进行了等位基因频率分析。gnomAD中c.2122G>A和c.2090G>A的等位基因频率极低(0.00089和0.00048),其他变异体未见报道。因此c.2450G>A、c.2122G>A和c.2090G>A变异体可能是致病的(图3中B)。
3为了明确IARS2变异的致病性,构建了来自患者2的永生化淋巴细胞。由于患者1身体原因,未能成功建立病人来源永生化淋巴细胞。与年龄匹配的对照细胞相比,患者2来源的永生化淋巴细胞中IARS2蛋白水平显著降低(图4中A)。进一步探究了患者来源永生化淋巴细胞中的OXPHOS复合物,结果表明,与对照细胞相比,复合物Ⅰ和Ⅲ的含量分别降低了约50%和约30%(图4中B)。与对照细胞相比,患者来源的永生化淋巴细胞显示出基础呼吸和ATP相关呼吸的显著降低(图5中A)。为了进一步探索IARS2变异对线粒体功能的影响,进一步检测了细胞ATP水平和MMP含量,结果显示与对照细胞相比,细胞ATP和MMP的产生减少(图5中B-C)。这些结果表明携带c.2450G>A和c.1_390del突变的患者来源永生化淋巴细胞线粒体功能受损。
4为了验证IARS2缺陷的致病性,基于shRNA构建了IARS2敲低的293T细胞,IARS2蛋白表达降低了80%(图6中A)。与患者来源永生化淋巴细胞一致,与对照细胞相比,IARS2敲低细胞中OXPHOS复合物Ⅰ和Ⅲ的含量显著降低(图6中B)。与对照细胞相比,在IARS2敲低细胞中基础氧呼吸和ATP依赖的氧呼吸也显著受损(图7中A)。此外,与对照细胞相比,IARS2敲低细胞中细胞ATP和MMP产生显著降低(图7中B-C)。这些结果表明IARS2对线粒体功能的维持是不可或缺的,且IARS2缺陷通过降低复合物Ⅰ和复合物Ⅲ含量导致线粒体功能障碍。
5为了进一步验证这些IARS2变异的致病性,在IARS2敲低的细胞中再表达了野生型IARS2和携带c.1_390del、c.2090G>A、c.2122G>A和c.2450G>A变异的IARS2。在IARS2敲低细胞中再表达野生型IARS2显著增加了IARS2蛋白水平。然而,相比野生型IARS2,再表达突变IARS2细胞中IARS2蛋白水平显著下降,其中c.1_390del变异降低最为明显(图8中A-D),表明c.1_390del、c.2090G>A、c.2122G>A和c.2450G>A变异均可以引起IARS2蛋白缺陷,表明c.1_390del、c.2090G>A、c.2122G>A和c.2450G>A变异均为致病性变异。如图9中A-B和图10中A-B所示,与再表达野生型IARS2的293T敲低细胞相比,再表达突变IARS2细胞中OXPHOS复合物Ⅰ和Ⅲ的含量、基础氧呼吸、ATP依赖氧呼吸及细胞ATP含量都显著降低。这些结果表明IARS2 c.2090G>A、c.2122G>A、c.2450G>A和c.1_390del能够导致线粒体功能障碍。
综上,本发明发现了IARS2基因缺陷导致线粒体病发生的分子机制,并验证了4个新突变位点(c.1_390del、c.2090G>A、c.2122G>A和c.2450G>A)具有致病性,拓展了IARS2基因突变谱,为IARS2缺陷相关线粒体病的临床诊断和预测提供了重要参考。
以上所述的实施例仅是对本发明的优选方式进行描述,并非对本发明的范围进行限定,在不脱离本发明设计精神的前提下,本领域普通技术人员对本发明的技术方案做出的各种变形和改进,均应落入本发明权利要求书确定的保护范围内。
Claims (6)
1.线粒体病相关IARS2基因突变位点,其特征在于,所述IARS2基因突变位点包括c.1_390del、c.2090G>A、c.2122G>A和c.2450G>A中的一种或多种;
所述c.2090G>A、c.2122G>A和c.2450G>A突变位点依次位于IARS2参考转录组序列的第2090、2122和2450位碱基处;其中,所述c.2090G>A、c.2122G>A和c.2450G>A突变位点碱基均突变为A;
所述c.1_390del突变位点的缺失片段位于IARS2参考转录组序列的第1-390位碱基处;
所述IARS2参考转录组序列登录号为NM_018060.4。
2.一种用于检测权利要求1所述IARS2基因突变位点的试剂在制备IARS2基因缺陷相关线粒体病辅助诊断或预测试剂盒中的应用。
3.根据权利要求2所述的应用,其特征在于,所述试剂包含引物组。
4.根据权利要求3所述的应用,其特征在于,所述引物组包括:
检测所述c.2090G>A、c.2122G>A和c.2450G>A突变位点的如SEQ ID NO:1-2所示的引物;检测所述c.1_390del突变位点的如SEQ ID NO:3-4所示的引物。
5.根据权利要求4所述的应用,其特征在于,若所述c.2090G>A、c.2122G>A和c.2450G>A突变位点均突变为A,和/或所述c.1_390del突变位点缺失目的片段,判断患者罹患IARS2基因缺陷相关线粒体病;
所述目的片段为IARS2参考转录组序列的第1-390位碱基片段;所述IARS2参考转录组序列登录号为NM_018060.4。
6.一种辅助诊断和预测IARS2基因缺陷相关线粒体病的试剂盒,其特征在于,包含权利要求4中所述的引物组。
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