PRKCH-NOS2A通路基因与缺血性脑卒中的关联研究
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摘要
缺血性脑卒中（ischemic stroke，IS）是我国致残率和致死率最高的常见疾病之一。动脉粥样硬化（Atherosclerosis，AS）是大多数缺血性脑卒中的病变基础,越来越多的证据表明AS的发生、进展均与炎症机制有关，长期轻度炎症是缺血性脑卒中发生的一个危险因素。经检索传导通路数据库，我们发现PRKCH-NOS2A通路基因与动脉内皮炎症反应相关。PRKCH基因是NOS2A基因上游的调控基因，由这一基因编码的蛋白激酶Cη（protein kinase Cη, PKCη）在粥样斑块的泡沫巨噬细胞和平滑肌细胞均中有表达，并且随着动脉粥样硬化程度的加重表达增加；而NOS2A基因编码的诱导型一氧化氮合酶（iNOS）是由巨噬细胞释放的重要的炎症介质之一，其表达可促发动脉内皮的炎症反应，导致AS形成及粥样斑块不稳定。因此，我们选择PRKCH-NOS2A通路基因作为缺血性脑卒中易感基因筛查的候选通路，以探讨PRKCH-NOS2A通路基因在缺血性脑卒中发病过程中的作用，阐明其在IS中的遗传学机制。
本研究选取吉林大学第一医院神经内科2009-2010年的住院患者505名作为病例组，经神经系统检查、头颅CT或头颅MRI明确脑卒中诊断；对照组来自同时期吉林大学第一医院体检中心的健康体检者，共505例。采集病例组及对照组入选人员全血用于DNA提取。通过阅读文献，浏览dbSNP数据库的人类基因组数据和PRKCH-NOS2A通路基因的SNPs图，发现PRKCH第9外显子、NOS2A基因启动子区及第1外显子可能与缺血性脑卒中的发生有关，我们应用基因测序的方法分别对这2个基因片段进行了测序。
在对PRKCH基因第9外显子进行测序的过程中，除1425G/A（rs2230500）和1427A/C（rs2230501）两个位点外，我们还发现另外3个SNP位点：rs57887308、rs58360439和rs17098394；通过对NOS2A基因启动子区及第1外显子的测序，发现-969G/C，-1173C/T（rs9282799）不是中国北方汉族人的SNP位点，NOS2A-969C位点的等位基因均为T，NOS2A-1173C位点的等位基因均为G，但我们在这一基因片段中发现另外3个SNP位点：-277T/C（rs2779248）、-1026C/A（rs2779249）和38C/G（rs10459953）。关联分析表明上述8个SNPs位点等位基因频率及基因型频率在缺血性脑卒中组与对照组之间无显著性差异（P>0.05）。我们依据A-S-C-O分型结合各项血管检查结果将缺血性脑卒中组进一步划分为大动脉粥样硬化性脑卒中组和小动脉闭塞性脑卒中组2组，关联分析表明各卒中亚组与对照组SNPs位点的基因频率及基因型频率仍无显著性差异（P>0.05）。此外，我们还对PRKCH-NOS2A通路基因与传统的卒中危险因素进行了数量性状分析，发现NOS2A基因SNP8（rs10459953）位点C等位基因与血浆LDL-c水平升高的协同作用与缺血性脑卒中相关（P=0.009），推测人群中C等位基因的携带者可能具有较高的iNOS表达水平，并与高LDL血症协同作用导致血管内皮功能障碍、加重炎症反应，引起动脉粥样硬化的发生，增加缺血性脑卒中发病风险。
综上，我们得出如下结论：①PRKCH基因rs2230500位点、rs2230501位点、rs57887038位点、rs58360439位点和rs17098394位点多态性可能与中国北方汉族人缺血性脑卒中无相关性；②NOS2A基因rs2779248位点、rs2779249位点和rs10459953位点多态性可能与中国北方汉族人缺血性脑卒中无相关性；③NOS2A基因rs10459953多态性与血浆LDL-c升高的协同作用与缺血性脑卒中相关。
本研究依据A-S-C-O分型标准结合多种血管检查方法划分卒中亚组，解决了以往应用TOAST分型划分卒中亚组时存在的不同医生对同一病例分型判断的一致性欠佳、病因分型比例出现偏移等问题，研究结果更为可靠；我们还对候选通路基因与脑卒中传统危险因素与缺血性脑卒中的关系进行了数量性状分析，更能够体现缺血性脑卒中多基因遗传疾病的发病本质；此外我们采用的基因测序方法也是目前分子遗传学领域的热点研究方法，通过一次基因检测可以同时获取大量的SNPs信息，操作简单、快捷，对于缺血性脑卒中这类多基因疾病候选基因的筛查更据优势。
Ischemic stroke (IS), a common neurological disease, is one of the leading causes ofsevere disability and death. Recently, accumulating evidence supports that atherosclerosis(AS) plays an important role in the development of IS, and that the disruption of the fibrouscap of vulnerable atheromatous plaques leads to intravascular thrombosis and acutecerebrovascular events. It is known that atherosclerosis is a chronic vascular inflammatoryprocess with the participation of several inflammatory cells and pathways. By retrieving thepathway database, we find the PRKCH-NOS2A pathway genes play a role in arterialendothelial inflammation. PRKCH gene, located in the upper sequence of NOS2A, is aregulator of NOS2A. This gene also encodes the protein kinase C η(PKC η), which isexpressed in the foam macrophages and smooth muscle cells of AS plaque. And induciblenitric oxide synthase (iNOS), coding by NOS2A gene, is a significant inflammatory factorwhich is released by macrophages and is responsible for endothelial inflammation. Therefore,we choose the PRKCH-NOS2A pathway as a candidate gene to discuss its relationship withischemic stroke.
We recruited505unrelated patients with ischemic stroke, admitted to the First Hospitalof Jilin University, Changchun, China in the period between2009and2010for thecase-control study. They were diagnosed as having ischemic stroke based on strictneurological examination, brain CT/MRI, which meet the International Classification ofDiseases (NCHS2007). The505controls came from the physical examination center, theFirst Hospital of Jilin University, in the period between2009and2010. Through readingliterature, browsing dbSNP database of the human genome data and SNPs map of thePRKCH-NOS2A pathway genes, we found exon9of the PRKCH gene, promoter and exon1of the NOS2A gene might play a role in the pathogenesis of ischemic stroke. Sequencinganalysis of these two gene segments was applied to genotype SNPs of them.
We found3other SNPs, except1425G/A (rs2230500) and1427A/C (rs2230501), inexon9of the PRKCH gene, and they were rs57887308, rs58360439and rs17098394. Bysequencing the promoter and exon1of the NOS2A gene, we found both-969G/C and-1173C/T were not SNPs of Chinese Han descent, but we found another3variants in thisNOS2A gene fragment:-277T/C(rs2779248),-1026C/A(rs2779249) and 38C/G(rs10459953). According to A-S-C-O classification standard, the case group wasdivided into aortic atherosclerotic stroke and lacunar infarction two subgroups. But thecocaphase analysis showed no significant differences in allele frequency and genotypefrequency between IS group, subgroups and control group (P>0.05). But, in the quantitiveanalysis we found that NOS2A gene C allele of SNP8(rs10459953) together with higherplasma low-density lipoprotein cholesterol level (P=0.009) was associated with IS. Wespeculate that groups with C allele may have higher iNOS expression level. And thisover-expressed iNOS may cooperate with higher plasma LDL-c and cholesterol level tocause endothelial dysfunction, increase inflammatory reaction, aggravate the process of AS,and finally increase the risk of ischemic stroke.
The data of our study suggests:①P olymorphisms ofrs2230500, rs2230501,rs57887038, rs58360439and rs17098394of the PRKCH gene is not associated withischemic stroke in a northern Chinese Han population;②Polymorphisms of rs2779248,rs2779249and rs10459953of the NOS2A gene is not associated with ischemic stroke in anorthern Chinese Han population;③NOS2A gene C allele of SNP8(rs10459953) togetherwith higher plasma low-density lipoprotein cholesterol level (P=0.009) may be associatedwith IS.
Our study is based on A-S-C-O classification standard combined with multiple vesselexaminations to divide stroke subtypes, avoiding case classification judgment uniformity byTOAST classification. The results are more reliable. Quantitive analysis of traditional IS riskfactors and candidate pathway genes is also applied in this study to uncover the character ofpolygenic diseases. In addition, gene sequencing is also a hot method in molecular genetics.A mass of information of SNPs gets from this simple and quick method. For ischemic stroke,as a polygenic disease, gene sequencing gains more advantages in the candidate genescreening.
本研究选取吉林大学第一医院神经内科2009-2010年的住院患者505名作为病例组，经神经系统检查、头颅CT或头颅MRI明确脑卒中诊断；对照组来自同时期吉林大学第一医院体检中心的健康体检者，共505例。采集病例组及对照组入选人员全血用于DNA提取。通过阅读文献，浏览dbSNP数据库的人类基因组数据和PRKCH-NOS2A通路基因的SNPs图，发现PRKCH第9外显子、NOS2A基因启动子区及第1外显子可能与缺血性脑卒中的发生有关，我们应用基因测序的方法分别对这2个基因片段进行了测序。
在对PRKCH基因第9外显子进行测序的过程中，除1425G/A（rs2230500）和1427A/C（rs2230501）两个位点外，我们还发现另外3个SNP位点：rs57887308、rs58360439和rs17098394；通过对NOS2A基因启动子区及第1外显子的测序，发现-969G/C，-1173C/T（rs9282799）不是中国北方汉族人的SNP位点，NOS2A-969C位点的等位基因均为T，NOS2A-1173C位点的等位基因均为G，但我们在这一基因片段中发现另外3个SNP位点：-277T/C（rs2779248）、-1026C/A（rs2779249）和38C/G（rs10459953）。关联分析表明上述8个SNPs位点等位基因频率及基因型频率在缺血性脑卒中组与对照组之间无显著性差异（P>0.05）。我们依据A-S-C-O分型结合各项血管检查结果将缺血性脑卒中组进一步划分为大动脉粥样硬化性脑卒中组和小动脉闭塞性脑卒中组2组，关联分析表明各卒中亚组与对照组SNPs位点的基因频率及基因型频率仍无显著性差异（P>0.05）。此外，我们还对PRKCH-NOS2A通路基因与传统的卒中危险因素进行了数量性状分析，发现NOS2A基因SNP8（rs10459953）位点C等位基因与血浆LDL-c水平升高的协同作用与缺血性脑卒中相关（P=0.009），推测人群中C等位基因的携带者可能具有较高的iNOS表达水平，并与高LDL血症协同作用导致血管内皮功能障碍、加重炎症反应，引起动脉粥样硬化的发生，增加缺血性脑卒中发病风险。
综上，我们得出如下结论：①PRKCH基因rs2230500位点、rs2230501位点、rs57887038位点、rs58360439位点和rs17098394位点多态性可能与中国北方汉族人缺血性脑卒中无相关性；②NOS2A基因rs2779248位点、rs2779249位点和rs10459953位点多态性可能与中国北方汉族人缺血性脑卒中无相关性；③NOS2A基因rs10459953多态性与血浆LDL-c升高的协同作用与缺血性脑卒中相关。
本研究依据A-S-C-O分型标准结合多种血管检查方法划分卒中亚组，解决了以往应用TOAST分型划分卒中亚组时存在的不同医生对同一病例分型判断的一致性欠佳、病因分型比例出现偏移等问题，研究结果更为可靠；我们还对候选通路基因与脑卒中传统危险因素与缺血性脑卒中的关系进行了数量性状分析，更能够体现缺血性脑卒中多基因遗传疾病的发病本质；此外我们采用的基因测序方法也是目前分子遗传学领域的热点研究方法，通过一次基因检测可以同时获取大量的SNPs信息，操作简单、快捷，对于缺血性脑卒中这类多基因疾病候选基因的筛查更据优势。
Ischemic stroke (IS), a common neurological disease, is one of the leading causes ofsevere disability and death. Recently, accumulating evidence supports that atherosclerosis(AS) plays an important role in the development of IS, and that the disruption of the fibrouscap of vulnerable atheromatous plaques leads to intravascular thrombosis and acutecerebrovascular events. It is known that atherosclerosis is a chronic vascular inflammatoryprocess with the participation of several inflammatory cells and pathways. By retrieving thepathway database, we find the PRKCH-NOS2A pathway genes play a role in arterialendothelial inflammation. PRKCH gene, located in the upper sequence of NOS2A, is aregulator of NOS2A. This gene also encodes the protein kinase C η(PKC η), which isexpressed in the foam macrophages and smooth muscle cells of AS plaque. And induciblenitric oxide synthase (iNOS), coding by NOS2A gene, is a significant inflammatory factorwhich is released by macrophages and is responsible for endothelial inflammation. Therefore,we choose the PRKCH-NOS2A pathway as a candidate gene to discuss its relationship withischemic stroke.
We recruited505unrelated patients with ischemic stroke, admitted to the First Hospitalof Jilin University, Changchun, China in the period between2009and2010for thecase-control study. They were diagnosed as having ischemic stroke based on strictneurological examination, brain CT/MRI, which meet the International Classification ofDiseases (NCHS2007). The505controls came from the physical examination center, theFirst Hospital of Jilin University, in the period between2009and2010. Through readingliterature, browsing dbSNP database of the human genome data and SNPs map of thePRKCH-NOS2A pathway genes, we found exon9of the PRKCH gene, promoter and exon1of the NOS2A gene might play a role in the pathogenesis of ischemic stroke. Sequencinganalysis of these two gene segments was applied to genotype SNPs of them.
We found3other SNPs, except1425G/A (rs2230500) and1427A/C (rs2230501), inexon9of the PRKCH gene, and they were rs57887308, rs58360439and rs17098394. Bysequencing the promoter and exon1of the NOS2A gene, we found both-969G/C and-1173C/T were not SNPs of Chinese Han descent, but we found another3variants in thisNOS2A gene fragment:-277T/C(rs2779248),-1026C/A(rs2779249) and 38C/G(rs10459953). According to A-S-C-O classification standard, the case group wasdivided into aortic atherosclerotic stroke and lacunar infarction two subgroups. But thecocaphase analysis showed no significant differences in allele frequency and genotypefrequency between IS group, subgroups and control group (P>0.05). But, in the quantitiveanalysis we found that NOS2A gene C allele of SNP8(rs10459953) together with higherplasma low-density lipoprotein cholesterol level (P=0.009) was associated with IS. Wespeculate that groups with C allele may have higher iNOS expression level. And thisover-expressed iNOS may cooperate with higher plasma LDL-c and cholesterol level tocause endothelial dysfunction, increase inflammatory reaction, aggravate the process of AS,and finally increase the risk of ischemic stroke.
The data of our study suggests:①P olymorphisms ofrs2230500, rs2230501,rs57887038, rs58360439and rs17098394of the PRKCH gene is not associated withischemic stroke in a northern Chinese Han population;②Polymorphisms of rs2779248,rs2779249and rs10459953of the NOS2A gene is not associated with ischemic stroke in anorthern Chinese Han population;③NOS2A gene C allele of SNP8(rs10459953) togetherwith higher plasma low-density lipoprotein cholesterol level (P=0.009) may be associatedwith IS.
Our study is based on A-S-C-O classification standard combined with multiple vesselexaminations to divide stroke subtypes, avoiding case classification judgment uniformity byTOAST classification. The results are more reliable. Quantitive analysis of traditional IS riskfactors and candidate pathway genes is also applied in this study to uncover the character ofpolygenic diseases. In addition, gene sequencing is also a hot method in molecular genetics.A mass of information of SNPs gets from this simple and quick method. For ischemic stroke,as a polygenic disease, gene sequencing gains more advantages in the candidate genescreening.
引文
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