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学科主题: 内科学
题名:
早发心肌梗死致病基因的筛查及功能研究
作者: 高元丰
答辩日期: 2016-05-17
导师: 陈红
专业: 内科学
授予单位: 北京大学
授予地点: 北京大学第二临床医学院
学位: 博士
关键词: 早发心肌梗死 ; 外显子组测序 ; CRISPR-Cas9技术 ; 基因突变
其他题名: Gene Screening and Functional Study on the Causative Genes of Premature Myocardial Infarction
分类号: R542.2
摘要:

背景:

心肌梗死(Myocardial Infarction,MI)是冠心病致死的主要原因。近年来,对急性心肌梗死及其危险因素的治疗和防控手段有了很大的发展,但是其死亡率依然高居不下。心肌梗死作为一种复杂疾病,其发生发展除受环境因素影响外,遗传因素亦为重要的病因。心肌梗死的双生子研究结果显示其遗传度(Heritability)可高达40%~63%,其中早发心肌梗死的遗传度(63%)更是明显高于非早发心肌梗死(40%),因此早发心肌梗死人群多用来心肌梗死遗传学研究的对象。既往心肌梗死的研究手段多集中于候选基因研究、全基因组关联研究(Genome-wide association study,GWAS)等。尤其是基于基因芯片技术的GWAS,其通过涉及数十万个冠心病患者及健康对照的大型合作研究,已发现了近60个与冠心病或心肌梗死相关联的基因区域,涉及近百个致病基因。但是,这些突变基因仅部分解释了心肌梗死的遗传学基质,尚有大量与疾病相关的致病基因有待发现。随着新一代测序技术(Next generation sequencing,NGS)的出现及不断完善,使得遗传病的研究更为有效和便捷。尤其是对于家系研究,由于同一家系内患病者的遗传同质性明显高于散发患者,利用新一代测序技术仅对家系内少数几个患病者进行直接测序以发现致病基因成为可能。

本研究组前期发现一早发心肌梗死家系,且其中多个成员伴有严重高胆固醇血症。本研究拟结合新一代测序技术及家系研究设计,试图进一步揭示早发心肌梗死的遗传学机制。

目的:

探索早发心肌梗死的新致病基因。

方法:

以确诊早发心肌梗死的两名患者为线索,在取得受试家系成员(n=17)的知情同意后,使用EDTA抗凝血管采取其4ml外周静脉血,并进一步询问病史、详细记录其临床信息及实验室检查结果,通过冠脉造影、冠状动脉增强CT及超声心动图等影像学手段明确家系成员冠状动脉病变情况。

1.Sanger测序

根据NCBI标准mRNA序列(NM_000527.4)利用Primer3软件进行引物设计,以覆盖LDLR(Low density lipoprotein receptor)基因全部外显子及外显子-内含子交界区域。使用Sanger测序法进行直接测序,完成对先证者外周血基因组DNA的LDLR基因进行基因筛查。

2.外显子组测序

对患者外周血基因组DNA行全外显子组测序(由诺禾致源公司完成)。将下述变异:(1)非基因区域变异、(2)正常SNPs、(3)同义突变、(4)功能预测为非致病,排除后,选取两名早发心肌梗死患者共同携带的变异,通过进一步的生物信息学分析及文献检索,初步选定一个或多个潜在致病突变。对发现的潜在致病突变位点,设计相应引物经Sanger测序验证所发现突变位点的确实性;其后,再利用同样方法,获得全部家系成员中该基因位点的突变情况,以观察所发现的突变位点在家系内的分布情况。进一步入选77例早发心肌梗死患者,取得知情同意的情况下使用EDTA抗凝血管采取其4ml外周血,询问并获得其临床信息。设计相应引物将所发现突变基因的全部外显子进行Sanger法直接测序,以明确所发现的致病基因在散发患者中发生突变的情况。基因测序结果的比对以及命名以NCBI上mRNA标准序列为参照。

利用Crispr-Cas9技术选取模式细胞进行基因修饰,在相应基因上引入所发现突变。通关相应基因的功能研究,以验证突变是否引起了基因功能的变化及其引起心肌梗死发生的潜在机制。

结果:

通过收集、分析该家系全部成员的临床信息,确定除了两名先证者外,另有其三叔其病史提示发生过典型心肌梗死症状,心电图V1-V6存在病理性q波且超声心动图检查提示室壁节段性运动异常,提示陈旧性心肌梗死,根据病史确定其心肌梗死发病年龄为53岁。另外,先证者同时患有高血压及高胆固醇血症,另一早发心肌梗死患者亦患有高血压;同时,其父方兄弟四人存在明显的高胆固醇血症,其母亲及长兄患有高血压。

1.致病基因的筛查

LDLR基因筛查发现,该家系中有六人为LDLR突变(c.1432G>A,p.Gly478Arg)携带者。但是,早发心肌梗死患者III.3并非携带者,且除先证者外,另外5名携带者均非早发心肌梗死患者,且其中4名携带者的冠状动脉增强CT结果提示其冠状动脉狭窄程度均<50%。

外显子组测序结果显示,两名早发心肌梗死患者共同携带的变异有27个,通过进一步的生物信息学分析(Phenolyzer基因型-表型分析器)及文献查阅,初步将SCAP-p.Ala1012Val及AGXT2-p.Ala338Val确定为潜在致病基因突变。对该家系内全部成员进行一代测序的结果显示,仅有两名早发心肌梗死患者同时携带两个突变,其余非早发心肌梗死家庭成员的仅携带≤1个上述突变。

2. 人群验证

进一步的人群验证结果显示,在77名早发心肌梗死患者中,发现另一名早发心肌梗死患者亦携带SCAP致病突变(c.1403T>C,p.Val468Ala),该变异在28,000名东亚人群中为阴性,且位于SCAP基因第468位的缬氨酸在物种间高度保守,提示其致病性。但并未发现AGXT2突变携带者。

3. 功能验证

我们上述两个潜在致病基因进行了相应细胞水平的功能研究。利用Crispr-Cas9技术对HH293T引入SCAP-p.Ala1012Val突变后,当培养基中胆固醇浓度超过50uM时,细胞核中成熟SREBP-2的表达量并未发生显著改变,而野生型HH293T细胞核中成熟SREBP-2的表达量则显著降低。使用胆固醇合成的直接前体角鲨烯20uM共培养24h亦可观察到上述现象。这提示SCAP-p.Ala1012Val突变可造成SCAP感受细胞内胆固醇浓度的能力下降。同时,我们使用Crispr-Cas9技术在EA.hy926细胞引入了AGXT2-p.Ala338Val突变,突变型EA.hy926相较野生型AGXT2细胞,其细胞上清液中ADMA水平显著升高,提示AGXT2编码蛋白的氨基转移酶功能降低。这些都提示该家系中发现的基因突变可导致相应基因功能异常,进而发挥潜在的致心肌梗死作用。

结论:

LDLR基因突变及严重高胆固醇血症并非引起心肌梗死的充分条件。SCAP与AGXT2是心肌梗死的潜在致病基因,二者发生基因突变时可分别通过影响细胞内胆固醇的负反馈调节机制及细胞内ADMA的代谢水平,从而发挥潜在的致心肌梗死作用。

英文摘要:

Bacground: Myocardial Infarction (MI) is the main cause of death in coronary heart disease (CHD). The prevention and treatments of MI have been involved during the past decades, the death rate stays high though. As a complex disease, genetic factor, the extent of which is defined by heritability, exerts a major influence on the cause of MI, as well as environment factors. Especially the premature MI, its heritability (63%) is significantly higher than than normal MI as seen from the results of twin study on MI. So it is more suitable for genetic study. Candidate gene studies and Genome-wide association study (GWAS) have been the most frequently utilized measures. Especially the chip-based GWAS, more than 60 MI or CHD related locus have been discovered by them, involving tens of thousands of patients and controls. However, only a small part of the genetic factors was explained, leaving most of the MI related genes undiscovered. With the arrival and ever-evolving of Next generation sequencing,(NGS), genetic study has been enormously facilitated, which could be more productive when accompanied by family-based study.

Objective: To investigate the causative genes of myocardial infarction (MI) in a pedigree with multiple members with premature myocardial infarction (PMI) by whole-exome sequencing and functional verification.

Methods: PMI was defined by MI diagnosed before the age of 50 for men and 60 for women. Peripheral blood of all the family members (n=17) was obtained under informed consent for DNA extraction. Sanger sequencing was utilized to define the mutation statues of LDLR in the family members of the pedigree.

Whole-exome sequencing was performed on 7 individuals from a PMI pedigree with two siblings diagnosed with MI before the age of 45 as well as their uncle at the age of 53. Bioinformatics analysis served as a tool for causative gene screening. Another 77 PMI patients were also enrolled for population validation of the potential causative genes. Finally, the potential causative genes were introduced into corresponding model cell lines by CRISPR-Cas9 technology, after which the functional consequences of the mutations were evaluated by western-blot and enzyme-linked immune-sorbent assay (Elisa).

Results: Sanger sequencing revealed 6 LDLR mutation carriers (c.1432G>A,p.Gly478Arg), of which only one was diagnosed with PMI, while the other five carriers not. Upon exome-sequencing and subsequent bioinformatics analysis, two variants in SCAP (c.3035C>T, p.Ala1012Val) and AGXT2 (c.1103C>T, p.Ala338Val) were identified as potential causative mutation for PMI. It was noteworthy that only patients that meet the diagnosis of PMI (n=2) harbored two variants all together, while other MI patients or members with no MI carried only one or no above variants.

We then screened the two genes in an independent population with PMI. Another variants was identified on SCAP (c.1403 T>C, p.Val468Ala), which was absent in 28, 000 east-Asian population. However, no potential causative mutation on AGXT2 was found in the present population.

Further, the two variants on SCAP and AGXT2 were introduced into HH293T and EA. hy926 cell lines respectively utilizing CRISPR-Cas9 measure. Functional study revealed that the SCAP mutation impaired SCAP-SREBP feedback mechanism which may lead to a “consititutive activation” effect of cholesterol synthesis related genes, while the AGXT2 mutation caused a deficiency of its aminotransferase activity which would lead to a down-regulation of NO production by ADMA accumulation.

Conclusion: SCAP and AGXT2 are potential causative genes for MI by impairing the feedback mechanism of cholesterol metabolism and ADMA metabolizing, respectively. Digenic mutation carriers may manifest a more severe phenotype, namely premature MI.

语种: 中文
相关网址: 查看原文
内容类型: 学位论文
URI标识: http://ir.bjmu.edu.cn/handle/400002259/124603
Appears in Collections:北京大学第二临床医学院_学位论文

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作者单位: 北京大学第二临床医学院

Recommended Citation:
高元丰. 早发心肌梗死致病基因的筛查及功能研究[D]. 北京大学第二临床医学院. 北京大学. 2016.
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