CN108588085B - 恶性心律失常的筛查试剂盒、动物模型的建立方法及应用 - Google Patents
恶性心律失常的筛查试剂盒、动物模型的建立方法及应用 Download PDFInfo
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Abstract
本发明提供了恶性心律失常的筛查试剂盒、动物模型的建立方法及应用。本发明在有家族性心源性猝死史的患者家系中检测到1个RyR2突变位点(p.A690E)并确认，进一步应用转基因动物模型，成功地构建了基因敲入小鼠，用小鼠模型验证了该突变可以导致猝死和室性心动过速的表型，还进一步应用生物化学、电生理、细胞和结构生物学等方法探讨了突变对钙通道结构和功能的影响，揭示了突变致儿茶酚胺敏感性多形性心室心动过速(CPVT)发病的分子机制。本发明为CPVT的治疗以及猝死的预防提供了新的思路，可应用小鼠模型筛选治疗心律失常的药物。
Description
技术领域
本发明涉及生物医学的疾病诊断技术领域，具体地说，涉及诱发恶性心律失常的RYR2基因突变位点的鉴定、检测、动物模型的建立及其在药物筛选中的应用。
背景技术
心源性猝死(sudden cardiac death，SCD)是指由于心脏原因所导致的突然死亡，它既可以发生于已知心脏病的患者中，也可发生在从无心脏病历史的健康人。心源性猝死常以突然丧失意识为表现。死亡通常在急性症状出现后1小时内发生，但是也有少数心脏骤停后存活者的死亡可超过此时限。在发达国家，心源性猝死是突然自然死亡的最常见原因。在美国，与心血管疾病有关的死亡大约占死亡人口总数的三分之一，而心源性猝死约占心血管死亡率的30％～40％。在中国，心血管疾病的发病率目前尚低于西方发达国家，但近些年来发病率上升很快。根据2009年发表的一项中国心源性猝死的流行病学调查报道，心源性猝死约占总死亡的9.5％，发生率约为41.8例/10万人口。如果以13亿人口推算，在中国心源性猝死的人数每年应高达54万。
大多数的心源性猝死发病于既有心血管病史的病患中，尸体解剖可见心血管结构异常(肥厚型心肌病、冠状动脉异常、心肌炎等)，然而一部分心源性猝死的尸检并未发现心血管有结构性异常，因此通常归类为原因不明性猝死。这一现象在国内的心源性猝死统计调查中也有发现，将近半数心源性猝死病例(43.5％的男性和42.3％的女性)生前并无明确心血管疾病病史(高血压、心绞痛、心肌梗死和心力衰竭等)。应用分子遗传学的方法分析原因不明性猝死，发现这类心脏结构无异常的猝死和人类RYR2基因上的突变相关，每7例原因不明性猝死中就有1例与RYR2基因变异相关。
人RYR2基因编码2型ryanodine受体，这一受体是心肌细胞中胞浆肌质网膜上钙离子释放通道。钙离子是各种细胞信号通路中最为重要的第二信使。在心脏中，钙离子调节心肌细胞收缩、调节心率以及调节细胞生长发育。在心肌细胞的兴奋-收缩偶联中，RyR2发挥至关重要的角色：兴奋信号传导至神经肌肉节点时引起肌细胞质膜去极化，电位变化沿质膜传导入心肌细胞深处，激活RyR2使其钙离子通道开放，胞浆肌质网中的钙离子释放到肌质中，结合肌钙蛋白引起肌丝构象变化，拉动肌丝滑动使心肌发生收缩。当RyR2通道关闭，肌质中的钙离子通过肌质网上ATP依赖的钙泵转运回肌质网中，肌质中的钙离子浓度随之降低，钙离子从肌钙蛋白上解离，使肌丝恢复原来的构象，引起肌纤维的舒张。
人类RYR2基因上第一个突变发现于2001年，迄今为止，大约有超过150例基因突变位点被识别出与2类遗传性心源性猝死相关：一类是儿茶酚胺敏感性多形性心室心动过速(catecholaminergic polymorphic ventriculartachycardia，CPVT)，另一类是2型致心律失常型右心室发育不良(arrhythmogenic right ventricular dysplasia type 2，ARVD2)。CPVT是一种遗传性心律失常，患者常在运动、疲劳或精神紧张时出现心室性心律失常，多为双向性室速，并可恶化为室颤进而导致心源性猝死，患者心脏结构多无明显异常。CPVT是青年人中原因不明性猝死的一个重要病因。ARVD2是一种遗传性心肌病，其特征为右心室心肌纤维发生病理改变导致右心室心肌薄弱，致使右心室室性心动过速反复发生造成昏厥并最终导致猝死。作为遗传性疾病，二者都是常染色体显性遗传病。
研究证实RyR2遗传变异是CPVT的主要原因，先证者中约有67％存在RyR2的突变。CPVT的分子和细胞水平致病机制尚不十分明了。至于基因突变所引起的RyR2结构异常以及如何造成其功能的紊乱进而诱发心律失常以及心源性猝死更是不清楚。
《华中科技大学》2013年硕士论文“RYR2基因G1885E，G1886S位点多态性与不明原因心脏性猝死的相关性探索”，RYR2基因G1886S位点在我国汉族健康人群中的分布存在多态性，且该位点与(Sudden unexplainedcaidiac death，SUD)SUD的发生具有明显的相关性，其中野生型G等位基因可能为SUD的保护基因，而突变型A等位基因可能是SUD的易感基因。
专利文献CN101500576A，公开日2009.08.05，公开了一种诊断受试者疾病或障碍的方法，该方法包含：从受试者获得细胞或组织样品；从细胞或组织中获得DNA；比较来自细胞或组织的DNA与编码RyR的对照核酸，以便确定来自细胞或组织的DNA中是否存在突变，突变的存在表示疾病或障碍。所述的突变为存在于编码RyR2的DNA中的突变。所述的RyR2突变位于染色体1q42-q43上。所述的突变为CPTV突变。
然而目前未见RYR2基因中A690E突变位点与心源性猝死相关关系的报道。
发明内容
本发明发现了一个新的RyR2突变位点(p.A690E)，其导致恶性心律失常。
第一方面，本发明提供了一种分离的编码突变体的核酸分子或其片段，所述的核酸分子或其片段为RYR2基因或其片段，且与SEQ ID NO:3相比，具有以下突变：c.142G>T突变。
第二方面，本发明提供了一种分离的多肽或其片段，所述的多肽或其片段为RyR2多肽或其片段，其序列与SEQ ID NO:4相比，具有p.A690E突变。
第三方面，本发明提供了一种用于筛查恶性心律失常或筛选易患恶性心律失常生物样品的试剂盒，含有：适于检测RYR2基因突变体的试剂，其中与SEQ ID NO:3相比，所述的RYR2基因突变体具有以下突变：c.142G>T突变。
作为一个优选例，所述的恶性心律失常选自儿茶酚胺敏感性多形性心室心动过速；房性和室性心律失常；心房和心室纤维性颤动；房性和室性快速型心律失常；房性和室性心动过速；及其运动诱发的变化形式。
作为另一优选例，所述的恶性心律失常为儿茶酚胺敏感性多形性心室心动过速。
第四方面，本发明提供了一种筛查恶性心律失常或筛选易患恶性心律失常生物样品的系统，包括：
核酸提取装置，所述的核酸提取装置用于从所述的生物样品提取核酸样本；
核酸序列确定装置，所述的核酸序列确定装置与所述的核酸提取装置相连，用于对所述的核酸样本进行分析，以便确定所述的核酸样本的核酸序列；
判断装置，所述的判断装置与所述的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SEQ ID NO:3相比，是否具有以下突变：c.142G>T突变。
第五方面，本发明提供了一种恶性心律失常的动物模型的建立方法，所述的建立方法是建立具有RyR2蛋白p.A690E突变位点的动物模型。
作为一个优选例，所述的动物模型选自大鼠、小鼠、狗、兔和猴。
作为另一优选例，采用CRISPR/Cas9方法建立恶性心律失常的动物模型。
第六方面，本发明提供了由所述建立方法所建立的动物模型在筛选治疗恶性心律失常的药物中的应用。
本文中，所述的“核酸分子”的类型不受特别限制，可以是任何包含与突变体的编码基因相对应的脱氧核糖核苷酸和/或核糖核苷酸的聚合物，包括但不限于DNA、RNA或cDNA。所述核酸，本领域技术人员应当理解，实际包括互补双链的任意一条，或者两条。为了方便，在本说明书和权利要求书中，虽然多数情况下只给出了一条链，但实际上也公开了与之互补的另一条链。例如，提及SEQ ID NO:3，实际包括其互补序列。本领域技术人员还可以理解，利用一条链可以检测另一条链，反之亦然。
所述的“生物样品”的类型并不受特别限制，只要从该生物样品中能够提取到反映生物样品是否存在突变的核酸样本即可。根据本发明的实施例，生物样品可以为选自人体血液、皮肤、皮下组织的至少一种，优选外周血。由此，可以方便地进行取样和检测，从而能够进一步提高筛选易患DCM的生物样品的效率。根据本发明的实施例，这里所使用的术语“核酸样本”应做广义理解，其可以是任何能够反映生物样品中是否存在突变的样本，例如可以是从生物样品中直接提取的全基因组DNA，也可以是该全基因组中包含致病基因编码序列的一部分，可以是从生物样品中提取的总RNA，也可以是从生物样品中提取的mRNA。
所述的“适于检测基因突变体的试剂”应做广义理解，即可以是检测病基因编码基因的试剂，也可以是检测突变体多肽的试剂，例如可以采用识别特异性位点的抗体。
本发明优点在于：
本发明的课题组重点致力于研究遗传突变位点在钙通道蛋白三维结构中的定位以及突变对蛋白结构、构象变化和功能的影响。在开展与儿茶酚胺敏感性多形性心室心动过速相关的遗传谱系调查以及致病基因的筛选时，在有家族性心源性猝死史的患者家系中检测到1个RYR2突变位点并进行确认，此位点以前从未被报道过。进一步应用转基因动物模型，成功地构建了基因敲入小鼠，并用小鼠模型验证了该突变可以导致猝死和室性心动过速的表型。本发明还进一步应用生物化学、电生理、细胞和结构生物学等方法探讨了突变对钙通道结构和功能的影响，揭示了突变致CPVT发病的分子机制。本发明为CPVT的治疗以及猝死的预防提供了新的思路，可应用小鼠模型筛选治疗心律失常的药物。
附图说明
图1.患者的家系。
图2.遗传突变的筛选情况。
图3.先证者和健康对照者的PCR定点测序比对。
图4.构建携带RyR2-A690E的基因敲入小鼠的原理。
图5.野生型RYR2基因序列、供体DNA序列和向导RNA序列。
图6.测序鉴定的电泳图谱，左图是F0代小鼠RYR2基因测序鉴定的电泳图谱，右图是F1代小鼠RYR2基因测序鉴定的电泳图谱。
图7.杂合子、野生型和纯合子小鼠RYR2基因对应位点的核苷酸序列。
图8.基因敲入小鼠的猝死表型。
图9.突变对RyR2钙离子通道功能的影响。
图10.突变对RyR2钙离子通道功能影响的分子机制。
图11.人和小鼠心肌RyR2，人和兔骨骼肌RyR2的序列比对以及蛋白质二级结构分析。
具体实施方式
下面结合附图对本发明提供的具体实施方式作详细说明。若未特别指明，实施例中所采用的技术手段为本领域技术人员所熟知的常规手段，可以参照《分子克隆实验指南》第3版或者相关产品进行，所采用的试剂和产品也均为可商业获得的。未详细描述的各种过程和方法是本领域中公知的常规方法，所用试剂的来源、商品名以及有必要列出其组成成分者，均在首次出现时标明，其后所用相同试剂如无特殊说明，均以首次标明的内容相同。
实施例1
患者为一个56岁女性，入住上海市第十人民医院心内科。心电图检查出现典型的室性心动过速。调查患者家族患病史发现其祖父祖母为近亲结婚，父亲早年因心脏病死亡，具体死亡原因因年代久远没有记录。患者姐妹三人，患者是年长姐姐，三妹45岁时上班路上追赶公交车突发猝死。患者儿子19岁，大学一年级，打篮球时突发猝死。依据临床心电图表型以及家族遗传史，我们高度怀疑其患有CPVT。依据美国心脏学会指南推荐，对患者进行基因检查。我们抽取了患者的外周血，提取DNA样本，做全基因组测序，发现有数千个遗传突变。我们重点关注与心律失常相关的213个基因，共筛选出9个基因上10个可疑突变位点。对患者的二妹(有同样的临床症状)和5位健康人做这些位点的测序鉴定，基因检测结果表明RYR2基因中A690E突变位点是患者和她的二妹独有的，因此判断此突变为高度疑似致病突变，该突变位点之前从未报道过。患者的家系见图1，遗传突变的筛选情况见图2，先证者和健康对照者的PCR定点测序比对见图3。
实施例2
我们建立动物模型，对突变是否为CPVT致病突变进行验证。
我们使用CRISPR/Cas9技术将突变位点敲入小鼠RYR2基因中。主要方法如下：
1、Cas9mRNA使用mMESSAGE
T7Ultra Kit进行体外转录；GuideRNA使用MEGAshortscriptTM Kit进行体外转录，产物均使用MEGAclearTMKit纯化。
2、注射样品配制成100μl体系，样品注射到受精卵雄原核中，供体受精卵取自C57BL/6J。
3、F0代小鼠出生14天剪取鼠尾0.3cm，加400μl裂解液及40μl蛋白酶K(10mg/ml)，56℃消化过夜。离心取上清，两倍体积无水乙醇沉淀，75％乙醇洗涤，稍晾干后溶解于100μl纯水中，50℃烘箱助溶1小时。
4、以各基因组为模板使用TaKaRa Lataq DNAPolymerase扩增鉴定片段，引物为Ryr2(A690E)-test-f1(1.0)和Ryr2(A690E)-test-r1。可扩增出1kb条带。反应条件为94℃5min变性后，94℃40s，63℃40s，72℃60s，进行34个循环，72℃延伸2min，PCR体系参见TaKaRa说明书。1％琼脂糖凝胶电泳后割胶回收1kb条带，分别连接T载体，转化，每个平板挑取6个单克隆测序鉴定，引物为Ryr2(A690E)-test-f1(1.0)和Ryr2(A690E)-test-r1。选取阳性小鼠与野生型C57BL/6J交配。
5、F1代小鼠出生14天剪取鼠尾0.3cm，加400μl裂解液及40μl蛋白酶K(10mg/ml)，56℃消化过夜。离心取上清，两倍体积无水乙醇沉淀，75％乙醇洗涤，稍晾干后溶解于100μl纯水中，50℃烘箱助溶1小时。
6、以各基因组为模板使用TaKaRa Lataq DNAPolymerase扩增鉴定片段，可扩增出1kb条带。反应条件为94℃5min变性后，94℃40s，63℃40s，72℃60s，进行34个循环，72℃延伸2min，PCR体系参见TaKaRa说明书。1％琼脂糖凝胶电泳后割胶回收1kb条带，分别连接T载体，转化，每个平板挑取6个单克隆测序鉴定。
图4显示了构建携带RyR2-A690E的基因敲入小鼠的原理。图5是野生型RYR2基因外显子20的序列、供体DNA序列和向导RNA序列。图6为测序鉴定的电泳图谱，左图是F0代小鼠RYR2基因测序鉴定的电泳图谱，右图是F1代小鼠RYR2基因测序鉴定的电泳图谱。图7是杂合子、野生型和纯合子小鼠RYR2基因对应位点的核苷酸序列。
携带有突变的小鼠杂合子和纯合子与野生型在共同的SPF级洁净环境与相同的饲养条件下做存活曲线测定。在长达1年的生存观测中无论是杂合子还是纯合子都表现出猝死的表型，而且杂合子的死亡率比纯合子更高(图8)。
我们提取了小鼠的心脏组织做生物化学研究，其中[3H]标记的ryanodine结合实验方法如下：
1、在研钵下垫好干冰，研钵内用加入适量液氮和心脏组织，小心的将心脏研磨成颗粒。
2、在冰上向玻璃研磨器内加入组织和匀浆破膜缓冲液(137mM NaCl，25mM Tris，50mM HEPES(pH7.4)，1％CHAPS)将组织匀浆，置于冰上反应1小时。
3、通过在4℃以16,000rpm离心30分钟除去不溶物质后，将上清液用于ryanodine结合。
4、30μl全心脏匀浆，加入KCl(终浓度150mM)，Tris(25mM)，HEPES(50mM，pH 7.4)，DTT(2.5mM)，蛋白酶抑制剂混合物，5nM[3H]ryanodine和300μl不同浓度的CaCl2溶液(最终游离Ca 2+从pCa 9.89到pCa 4)，充分混合后，孵育37℃ 3小时。
5、先使用4度预冷的1％polyethylenimine溶液润湿GF/B玻璃微纤维滤纸，然后再滤过37℃孵育完毕的混合液，再用预冷的洗涤缓冲液洗涤结合混合物三次。
6、通过液体闪烁计数器检测放射性。
实验结果(图9)表明突变引起半数有效浓度降低，表明钙通道对钙离子浓度的敏感性增强，正常的细胞钙离子浓度不能激活正常钙通道但可以激活突变的钙通道，造成钙通道不正常的激活，进一步引发心肌收缩的异常，产生心律失常。
我们应用冷冻电镜技术测定了RyR2通道蛋白的三维结构。我们分析了A690E这一位点在RyR2三维结构中的精准定位，实验方法包括冷冻电镜三维结构测定、RyR2片段的晶体结构在冷冻电镜结构中的定位、突变位点的精准定位。我们的结构分析结果(图10)表明这一突变位点A690E位于一段称为SPRY1的结构域中(条带结构)，将SPRY1的结构配位进RyR全序列的冷冻电镜电子密度图中，发现其位于RyR2与FKBP的结合位点上。FKBP是RyR2的调控蛋白，FKBP与RyR2的结合可以稳定钙通道的稳定性，而FKBP的解离会减弱钙通道的稳定性，使钙通道易于激活而产生钙泄露。A690E的突变发生了氨基酸电荷的变化，即由不带电的A突变成带负电荷的D。在FKBP结合面上带有4个带负电荷的氨基酸，D38、D43、D101和E103。由于静电排斥作用，突变会减弱RyR2-FKBP的结合，造成钙通道稳定性的减弱。
以下为本发明的核苷酸和氨基酸序列说明：
1、用于检测RYR2基因中A690E突变位点的引物：
Ryr2(A690E)-test-f1(1.0)(SEQ ID NO:1)：
5'TGGGCGTCAGTGAAGGTTCT 3'
Ryr2(A690E)-test-r1(SEQ ID NO:2)：
5'TTCTGGAACACGGTGAAAGA 3'
2、健康人RYR2基因外显子20的序列(SEQ ID NO:3)(备注：测序方向为反向，下划线标示部分为突变位置)：
3、健康人RyR2的部分氨基酸序列(SEQ ID NO:4)(备注：人RYR2基因编码RyR2蛋白，一级序列全长4967个氨基酸，其序列可在NCBI网站查询https:// www.ncbi.nlm.nih.gov/protein/NP_001026.2，因蛋白全长过长，这里只列出包含有690位突变的结构域序列(652-844)，突变位点A690以下划线标出)：
人和小鼠心肌RyR2，人和兔骨骼肌RyR2的序列比对以及蛋白质二级结构分析见图11。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员，在不脱离本发明方法的前提下，还可以做出若干改进和补充，这些改进和补充也应视为本发明的保护范围。
SEQUENCE LISTING
<110> 上海市第十人民医院
<120> 恶性心律失常的筛查试剂盒、动物模型的建立方法及应用
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Claims (9)
1. 一种分离的编码突变体的核酸分子，其特征在于，所述的核酸分子为在SEQ ID NO:3 基础上第142位碱基由G突变为T 的核酸分子。
2.一种分离的多肽，其特征在于，所述的多肽为RyR2多肽，其序列与NCBI中编号为NP_001026.2的健康人RyR2蛋白序列相比，第690位的氨基酸残基由A突变为E。
3. 一种用于筛查恶性心律失常或筛选易患恶性心律失常生物样品的试剂盒，其特征在于，含有：适于检测RYR2 基因突变体的试剂，其中与SEQ ID NO:3相比，所述的RYR2 基因突变体的突变为：第142位碱基由G突变为T。
4. 根据权利要求3 所述的试剂盒，其特征在于，所述的恶性心律失常为儿茶酚胺敏感性多形性心室心动过速。
5.一种筛查恶性心律失常或筛选易患恶性心律失常生物样品的系统，其特征在于，包括：
核酸提取装置，所述的核酸提取装置用于从所述的生物样品提取核酸样本；
核酸序列确定装置，所述的核酸序列确定装置与所述的核酸提取装置相连，用于对所述的核酸样本进行分析，以便确定所述的核酸样本的核酸序列；
判断装置，所述的判断装置与所述的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SEQ ID NO:3 相比，判断是否发生第142位碱基由G突变为T。
6.一种恶性心律失常的动物模型的建立方法，其特征在于，建立RyR2蛋白在NCBI中编号NP_001026.2所示序列基础上发生了第690位氨基酸残基由A突变为E的动物模型，所述RyR2蛋白其序列如NCBI中编号NP_001026.2所示。
7. 根据权利要求6 所述的建立方法，其特征在于，所述的动物模型选自大鼠、小鼠、狗、兔和猴。
8. 根据权利要求6 所述的建立方法，其特征在于，采用CRISPR/Cas9 方法建立恶性心律失常的动物模型。
9. 由权利要求6 所述建立方法所建立的动物模型在筛选治疗恶性心律失常的药物中的应用。
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