PDE4D及IL-1基因多态性与中国汉族人群缺血性卒中的关联研究
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摘要
研究背景
脑血管疾病(Cerebrovascular Disease, CVD)作为一类常见病、多发病,严重威胁着人类的健康,是当今人类三大死亡原因之一。缺血性卒中(Ischemic Stroke, IS)约占全部CVD的70%。现认为IS是由遗传因素与环境因素相互作用所致的复杂性疾病,而遗传因素在其发病机制中发挥了尤为重要的作用。目前,应用关联分析技术对缺血性卒中的候选基因的筛查已经取得了一些研究成果,主要包括：①炎症系统相关基因；②脂代谢基因；③肾素-血管紧张素系统相关基因；④一氧化氮合酶基因。但由于上述基因在不同的国家、地区或种族之间能够得到较好重复性结果的并不多,提示这类研究可能具有较大的地域性差异,因此需要在不同的人群中继续进行验证。
动脉粥样硬化的形成和发展是IS的重要病理生理基础。目前已经证实,炎症反应在这一过程中具有极其重要的意义。因此,本研究着眼于IS发病率较高的中国北方汉族人群,选择了炎症系统调控基因中的重要组成部分一磷酸二酯酶(Phosphodiesterase, PDE) 4D与白细胞介素-1(Interleukin-1, IL-1)基因作为候选基因,采用病例-对照研究的方法,分析比较PDE4D基因多态性位点SNP83、SNP87、SNP219、SNP220和IL-1基因多态性位点IL-1A-88、IL-1B-511的基因型、等位基因型在病例组和对照组中频率分布的差异,进而探讨这两种基因多态性与中国北方汉族人群IS之间的关联,以期为更详尽地了解IS潜在的遗传发病机制提供一定的分子流行病学依据,并为早期诊断该病提供更多的分子生物学指标。
研究方法
采用病例-对照的研究方法,病例组按病因不同分为大血管亚组(大动脉粥样硬化型IS组,即血栓性脑梗死组)和小血管亚组(腔隙性脑梗死组,简称腔梗组)。大血管亚组选取中国北方汉族大动脉粥样硬化型IS患者371例,其中男230例,女141例,年龄在41～74岁间,平均年龄63.88±7.36岁,彼此无亲缘关系；小血管亚组选取中国北方汉族腔隙性脑梗死(简称腔梗)患者302例,年龄41～76岁,平均年龄61.42±9.40岁,男183例,女119例,彼此无亲缘关系。选择性别、年龄与病例组相匹配的健康个体371例作为对照组,其中男247例、女124例；应用聚合酶链式反应-限制性片段长度多态性(Polymerase Chain Reaction---estriction Fragment Length Polymorphism Technique,PCR-RFLP)的技术检测病例组和对照组PDE4D基因SNP83、SNP87位点和IL-1A-889、IL-1B-511位点的多态性,应用基质辅助激光解吸附电离飞行时间质谱引物延伸(Matrix-assisted Laser Desorption/ Ionization Time-of-flight Mass Spectrometry Primer Extension, MALDI-TOF-PEX)技术检测大血管亚组和对照组PDE4D基因SNP219、SNP220位点多态性；基因型结果经双脱氧末端终止测序法进一步确认。应用拟合优度χ2检验来验证每个SNP位点基因型在抽样群体中的分布是否符合Hardy-Weinberg平衡。对于连续性变量,以均值±标准差表示,采用单因素方差分析进行检验。对于单个SNP位点的数据,通过SPSS13.0统计软件,以x2 Fisher's精确概率法检验其基因型、等位基因型在对照组与病例组中的频率分布差异、并评估风险度。之后结合临床资料,对相关数据进行条件Logistic回归分析,以排除传统混杂因素的干扰,P<0.05表示具有统计学差异。PDE4D与IL-1基因对IS的影响以优势比(Odds Ratio, OR)和95%可信区间(95% Confidence Interval,95%CI)来评估。应用SHEsis在线软件计算病例组和对照组PDE4D基因单体型的频率分布差异,P<0.05表示具有统计学差异。
研究结果
1、病例组和对照组PDE4D基因的每个多态性位点基因型的平衡检验结果表明它们的基因型频率分布没有偏离Hardy-Weinberg平衡(P>0.05)。表示所选取的样本具有人群代表性,适合进行遗传学分析。
2、PDE4D基因多态性在对照组与大血管亚组中的频率分布为：大血管亚组SNP83位点CC基因型和C等位基因型频率分布均高于对照组(CC:P=0.001;OR=1.788；95％CI:1.280-2.497；C:P=0.003；OR=1-368；95％CI:1.115-1.678).SNP 219位点AA基因型和A等位基因型频率高于对照组(AA:P=0.029；OR=1.444；95％CI: 1.037-2.010；A:P=0.048；OR=1.228；95％CI:1.001-1.505).经条件Logistic回归校正后,大血管亚组SNP83CC基因型分布频率仍然高于对照组(P=0.036；OR=1.603；95％CI:1.032～2.489),SNP219AA基因型分布频率也仍然高于对照组(P=0.046; OR=1.567；95％CI:1.008-2.436).
3.PDE4D基因多态性位点SNP83.SNP87在对照组与小血管亚组中的基因型、等位基因型频率分布未见显著性差异。
4.PDE4D基因单体型在对照组与大血管亚组中的频率分布为：由SNP83、SNP87位点构建的单体型频率分布在两组之间未见统计学差异。由SNP219、SNP220位点构建的单体型中,大血管亚组的AA单体型频率高于对照组(P=0.013；OR.296；95％CI:1.056-1.591)；而GA单体型频率病例组低于对照组,(P=0.005；OR=0.650；95％CI:0.482-0.878).
5.PDE4D基因单体型在对照组与小血管亚组中的频率分布为：由SNP83. SNP87位点构建的单体型中,对照组CT.TT单体型频率显著高于小血管亚组(CT: P=0.014；OR=0.637；95％CI:0.444～0.913；TT:P=0.004；OR=0.662；95％CI:0.528～0.830)。而小血管亚组TC单体型频率显著高于对照组(P=0.002；OR=1.861；95％CI: 1.391-2.490).
6、IL-1基因多态性位点的基因型和等位基因型在病例组与对照组中的频率分布比较：病例组IL-1A基因-889位点的T等位基因型频率与对照组相比具有显著统计学差异(P=0.024:OR=1.277:95％CI:1.033-1.577).经过条件logistic回归校正后,病例组T等位基因型频率与对照组相比较,仍存在统计学差异(P=0.034: OR.913:95％CI:1.621-2.375).IL-1B-511位点在病例、对照组之间各基因型、等位基因型频率分布未见统计学差异差异。
结论
1.PDE4D基因SNP83位点可能与中国北方汉族人群大血管性IS具有相关性,其风险性主要来自于C等位基因。
2、通过对大血管亚组PDE4D基因SNP83.SNP87.SNP219.SNP220位点的连锁不平衡分析,所研究的人群中并未发现由SNP83、SNP87位点构建的危险性或保护性单体型,而在由SNP219、SNP220位点构建的单体型中,AA可能是危险性单体型,GA可能是保护性单体型。
3、PDE4D基因SNP83、SNP87位点组成的TC单体型在小血管亚组中的分布频率显著高于对照组,可能是一种能够增加腔梗患病风险的危险性单体型。
4、PDE4D基因SNP83、SNP87位点组成的CT、TT单体型在对照组中的频率分布显著高于小血管亚组,可能属于能够降低腔梗患病风险的保护性单体型。
5.PDE4D基因可能与中国北方汉族人群腔隙性脑梗死的患病风险具有一定的关联,但需要继续进行大样本检测。
6、支持deCODE研究组所做出的关于PDE4D基因可能是IS易感基因的结论,但具体的多态性位点可能存在地区、种族差异。
7、IL-1A基因-889位点T等位基因型可能与中国北方汉族人群大血管性IS之间具有一定的相关性,但是否为该病的易患基因尚有待更大样本的研究证实。
8、IL-1B-511位点基因多态性可能与中国北方汉族人群大血管性IS之间无显著的相关性。
9、IL-1基因多态性与IS之间的相关性强弱与否也可能受到地区、种族差异的影响。
Cerebrovascular disease(CVD) is one of the three major causes of death, and it is harmful to human health. Ischemic stroke(IS) accounts for about 70%of all CVD. IS is currently considered as a kind of complex disease influenced by the interaction of genetic and environmental factors, while genetic factors may play an important role in the pathogenesis. At present, the candidate genes of IS obtained by association analysis mainly include:①inflammation-related genes;②lipid metabolism genes;③renin angiotensin system-related genes;④nitric oxide synthase gene. However, these studies in different countries, regions or ethnic origin can get few good replication, suggesting that such research may have great regional differences. Therefore, it is necessary to verify those candidate genes in different populations.
Atherosclerosis is the important pathophysiological basis IS. Now, it has been confirmed that the inflammatory reaction was of great importance in the process of atherosclerosis. Therefore, this study based on the Han population in northern China, a region having high incidence of IS, and selected Phosphodiesterase 4D(PDE4D) and Interleukin-1(IL-1), two important components of genes regulating the inflammatory system. In this candidate genes case-control analysis, we mesured genotype and allele frequencies of four PDE4D gene's SNPs (SNP83、SNP87、SNP219、SNP220) and two IL-1 gene's SNPs (IL-1A-889、IL-1B-511) between case and control group, and evaluated the association between these two genes'SNPs and IS. Therefore, we may provide more molecular epidemiology data for comprehensively understanding the potential genetic mechanisms and more early molecular biological diagnosis indicators of IS.
Materials and methods
A case-control design was introduced. According to the different causes, the patients were divided into two subgroups:the large vessel group (thrombotic cerebral infarction group) and the small vessel group(lacunar infarction group).The subjects of large vessel group were consecutively recruited from the patients of northern China region patients with a total number of 371 cases, male 240, female 141, aged 41-74 years old, Han ethnic, no consanguinity with each other, the small vessel group were consecutively recruited from northern China region patients with a total number of 302 cases, male183,female 119, aged 41-76 years old, Han ethnic, no consanguinity with each other. The controls matched by age, sex, and ethnic orign were recruited from the same geographic region, with a total of 371, male 247, female 124. The SNPs of PDE4D SNP83、87 and IL-1A-889、IL-1B-511 were genotyped by the technique of polymerase chain reaction and restriction fragment length polymorphism(PCR-RFLP). PDE4D SNP219、220 were genotyped by means of Matrix-assisted laser desorption /ionization time-of-flight mass spectrometry primer extension (MALDI-TOF-PEX)reaction. Genotyping results were confirmed by dideoxy termination squencing method. SPSS 13.0 statistical software was used for data processing. Continuous variables were expressed as mean±SE and were assessed by One-Way ANOVA. Genotype and allele frequencies were calculated for control group and each of the patient groups. Univariate comparisons of allele and genotype distributions were analyzed by x2 test. The x2 goodness of fit test was used to test for deviation of genotype distribution from Hardy-Weinberg equilibrium. Conditional Logistic Regression was used for adjusting for IS traditional risk factors of IS. The influence of PDE4D and IL-1 gene polymorphisms on IS was indicated by odds ratio(OR) and 95% confidence interval(CI) with P<0.05 as significant criteria. Haplotype-based analysis was infered by using SHEsis online-software.
Results
1. The genotype frequencies of all SNPs conformed to the expectations of Hardy-Weinberg equilibrium(P>0.05). It was suggested that the selected samples could represent the population and were suitable for genetic analysis.
2. PDE4D gene SNPs frequencies distributions among the large vessel and control group:SNP83CC genotype and C allele frequencies of the case group were higher than the control group(CC:P=0.001; OR= 1.788; 95%CI:1.280-2.497;C:P=0.003; OR= 1.368;95%CI:1.115-1.678); SNP219 AA genotyp and A allele frequencies of the large vessels group were higher than the control group(P=0.029; OR= 1.444; 95%CI: 1.037-2.010; A:P=0.048; OR= 1.228;95%CI:1.001-1.505), and after conditional logistic regression the differece was still significant(SNP83:OR= 1.479; 95%CI: 1.024-2.135; P=0.037; SNP219:P=0.046; OR=1.567; 95%CI:1.008-2.436).
3. PDE4D gene SNPs'frequencies distributions among the small vessel and control group:Significant differences of all the frequencies were not observed between these two groups.
4.PDE4D gene haplotypes'frequencies among the large vessel and control group: The frequencies of the haplotype built by SNP83 and SNP87 were normal among the patients and the controls; Among the haplotypes built by SNP219 and SNP220, AA's frequency of the case group was higher than the control group(P=0.013;OR=1.296; 95%CI:1.056-1.591), while the frequency of GA in the case group was lower than the control group(P=0.005; OR=0.650; 95%CI:0.482-0.878).
5.PDE4D gene haplotypes'frequencies among the small vessel and control group: the frequencies of CT and TT in the control group were higher than those in the case group(CT:P=0.014;OR=0.637; 95%CI:0.444-0.913;TT:P=0.004; OR=0.662; 95%CI: 0.528-0.830),while the frequency of TC in the case group was higher than which in the control group(P=0.002;OR=1.861; 95%CI:1.391-2.490).
6.IL-1SNPs'frequencies distributions among the cases and controls:the T allele's frequency of IL-1A-889 of the case group was higher than the control group (P=0.024; OR= 1.277;95%CI:1.033-1.577), and after conditional logistic regression the difference was still significant(P=0.034; OR=1.913; 95%CI:1.621-2.375). All the frequencies of genotypes and alleles of IL-1B-511 between the two groups had no significant differences.
Conclusions
1. PDE4D SNP83 might be associated with the risk of thrombotic cerebral infarction in northern han Chinese.
2. The haplotypes built by PDE4D SNP83 and SNP87 might have no association With thrombotic cerebral infarction. The haplotype AA built by SNP219 and SNP220 might be at-risk haplotype of thrombotic cerebral infarction, meanwhile, GA might be be protective haplotype.
3.The haplotype TC built by PDE4D SNP83 and SNP87 might be at-risk haplotype of lacunar infarction.
4.The haplotype CT and TT built by PDE4D SNP83 and SNP87 might be protective haplotypes of lacunar infarction.
5.PDE4D gene might be associated with the risk of lacunar infarction in northern han Chinese.
6.Although t he result of our study supports the conclusion of deCODE, there still be some difference because of the difference of regions and ethnics.
7. IL-1A-889 T allele might be associated with thrombotic cerebral infarction in northern han Chinese, but it need to be tested by larger studies.
8. IL-1B-511 might have no association with IS in northern han Chinese.
9. The association between IL-1 polymorphism and IS might be affected by the difference of regions、ethnics and nationalities.
脑血管疾病(Cerebrovascular Disease, CVD)作为一类常见病、多发病,严重威胁着人类的健康,是当今人类三大死亡原因之一。缺血性卒中(Ischemic Stroke, IS)约占全部CVD的70%。现认为IS是由遗传因素与环境因素相互作用所致的复杂性疾病,而遗传因素在其发病机制中发挥了尤为重要的作用。目前,应用关联分析技术对缺血性卒中的候选基因的筛查已经取得了一些研究成果,主要包括：①炎症系统相关基因；②脂代谢基因；③肾素-血管紧张素系统相关基因；④一氧化氮合酶基因。但由于上述基因在不同的国家、地区或种族之间能够得到较好重复性结果的并不多,提示这类研究可能具有较大的地域性差异,因此需要在不同的人群中继续进行验证。
动脉粥样硬化的形成和发展是IS的重要病理生理基础。目前已经证实,炎症反应在这一过程中具有极其重要的意义。因此,本研究着眼于IS发病率较高的中国北方汉族人群,选择了炎症系统调控基因中的重要组成部分一磷酸二酯酶(Phosphodiesterase, PDE) 4D与白细胞介素-1(Interleukin-1, IL-1)基因作为候选基因,采用病例-对照研究的方法,分析比较PDE4D基因多态性位点SNP83、SNP87、SNP219、SNP220和IL-1基因多态性位点IL-1A-88、IL-1B-511的基因型、等位基因型在病例组和对照组中频率分布的差异,进而探讨这两种基因多态性与中国北方汉族人群IS之间的关联,以期为更详尽地了解IS潜在的遗传发病机制提供一定的分子流行病学依据,并为早期诊断该病提供更多的分子生物学指标。
研究方法
采用病例-对照的研究方法,病例组按病因不同分为大血管亚组(大动脉粥样硬化型IS组,即血栓性脑梗死组)和小血管亚组(腔隙性脑梗死组,简称腔梗组)。大血管亚组选取中国北方汉族大动脉粥样硬化型IS患者371例,其中男230例,女141例,年龄在41～74岁间,平均年龄63.88±7.36岁,彼此无亲缘关系；小血管亚组选取中国北方汉族腔隙性脑梗死(简称腔梗)患者302例,年龄41～76岁,平均年龄61.42±9.40岁,男183例,女119例,彼此无亲缘关系。选择性别、年龄与病例组相匹配的健康个体371例作为对照组,其中男247例、女124例；应用聚合酶链式反应-限制性片段长度多态性(Polymerase Chain Reaction---estriction Fragment Length Polymorphism Technique,PCR-RFLP)的技术检测病例组和对照组PDE4D基因SNP83、SNP87位点和IL-1A-889、IL-1B-511位点的多态性,应用基质辅助激光解吸附电离飞行时间质谱引物延伸(Matrix-assisted Laser Desorption/ Ionization Time-of-flight Mass Spectrometry Primer Extension, MALDI-TOF-PEX)技术检测大血管亚组和对照组PDE4D基因SNP219、SNP220位点多态性；基因型结果经双脱氧末端终止测序法进一步确认。应用拟合优度χ2检验来验证每个SNP位点基因型在抽样群体中的分布是否符合Hardy-Weinberg平衡。对于连续性变量,以均值±标准差表示,采用单因素方差分析进行检验。对于单个SNP位点的数据,通过SPSS13.0统计软件,以x2 Fisher's精确概率法检验其基因型、等位基因型在对照组与病例组中的频率分布差异、并评估风险度。之后结合临床资料,对相关数据进行条件Logistic回归分析,以排除传统混杂因素的干扰,P<0.05表示具有统计学差异。PDE4D与IL-1基因对IS的影响以优势比(Odds Ratio, OR)和95%可信区间(95% Confidence Interval,95%CI)来评估。应用SHEsis在线软件计算病例组和对照组PDE4D基因单体型的频率分布差异,P<0.05表示具有统计学差异。
研究结果
1、病例组和对照组PDE4D基因的每个多态性位点基因型的平衡检验结果表明它们的基因型频率分布没有偏离Hardy-Weinberg平衡(P>0.05)。表示所选取的样本具有人群代表性,适合进行遗传学分析。
2、PDE4D基因多态性在对照组与大血管亚组中的频率分布为：大血管亚组SNP83位点CC基因型和C等位基因型频率分布均高于对照组(CC:P=0.001;OR=1.788；95％CI:1.280-2.497；C:P=0.003；OR=1-368；95％CI:1.115-1.678).SNP 219位点AA基因型和A等位基因型频率高于对照组(AA:P=0.029；OR=1.444；95％CI: 1.037-2.010；A:P=0.048；OR=1.228；95％CI:1.001-1.505).经条件Logistic回归校正后,大血管亚组SNP83CC基因型分布频率仍然高于对照组(P=0.036；OR=1.603；95％CI:1.032～2.489),SNP219AA基因型分布频率也仍然高于对照组(P=0.046; OR=1.567；95％CI:1.008-2.436).
3.PDE4D基因多态性位点SNP83.SNP87在对照组与小血管亚组中的基因型、等位基因型频率分布未见显著性差异。
4.PDE4D基因单体型在对照组与大血管亚组中的频率分布为：由SNP83、SNP87位点构建的单体型频率分布在两组之间未见统计学差异。由SNP219、SNP220位点构建的单体型中,大血管亚组的AA单体型频率高于对照组(P=0.013；OR.296；95％CI:1.056-1.591)；而GA单体型频率病例组低于对照组,(P=0.005；OR=0.650；95％CI:0.482-0.878).
5.PDE4D基因单体型在对照组与小血管亚组中的频率分布为：由SNP83. SNP87位点构建的单体型中,对照组CT.TT单体型频率显著高于小血管亚组(CT: P=0.014；OR=0.637；95％CI:0.444～0.913；TT:P=0.004；OR=0.662；95％CI:0.528～0.830)。而小血管亚组TC单体型频率显著高于对照组(P=0.002；OR=1.861；95％CI: 1.391-2.490).
6、IL-1基因多态性位点的基因型和等位基因型在病例组与对照组中的频率分布比较：病例组IL-1A基因-889位点的T等位基因型频率与对照组相比具有显著统计学差异(P=0.024:OR=1.277:95％CI:1.033-1.577).经过条件logistic回归校正后,病例组T等位基因型频率与对照组相比较,仍存在统计学差异(P=0.034: OR.913:95％CI:1.621-2.375).IL-1B-511位点在病例、对照组之间各基因型、等位基因型频率分布未见统计学差异差异。
结论
1.PDE4D基因SNP83位点可能与中国北方汉族人群大血管性IS具有相关性,其风险性主要来自于C等位基因。
2、通过对大血管亚组PDE4D基因SNP83.SNP87.SNP219.SNP220位点的连锁不平衡分析,所研究的人群中并未发现由SNP83、SNP87位点构建的危险性或保护性单体型,而在由SNP219、SNP220位点构建的单体型中,AA可能是危险性单体型,GA可能是保护性单体型。
3、PDE4D基因SNP83、SNP87位点组成的TC单体型在小血管亚组中的分布频率显著高于对照组,可能是一种能够增加腔梗患病风险的危险性单体型。
4、PDE4D基因SNP83、SNP87位点组成的CT、TT单体型在对照组中的频率分布显著高于小血管亚组,可能属于能够降低腔梗患病风险的保护性单体型。
5.PDE4D基因可能与中国北方汉族人群腔隙性脑梗死的患病风险具有一定的关联,但需要继续进行大样本检测。
6、支持deCODE研究组所做出的关于PDE4D基因可能是IS易感基因的结论,但具体的多态性位点可能存在地区、种族差异。
7、IL-1A基因-889位点T等位基因型可能与中国北方汉族人群大血管性IS之间具有一定的相关性,但是否为该病的易患基因尚有待更大样本的研究证实。
8、IL-1B-511位点基因多态性可能与中国北方汉族人群大血管性IS之间无显著的相关性。
9、IL-1基因多态性与IS之间的相关性强弱与否也可能受到地区、种族差异的影响。
Cerebrovascular disease(CVD) is one of the three major causes of death, and it is harmful to human health. Ischemic stroke(IS) accounts for about 70%of all CVD. IS is currently considered as a kind of complex disease influenced by the interaction of genetic and environmental factors, while genetic factors may play an important role in the pathogenesis. At present, the candidate genes of IS obtained by association analysis mainly include:①inflammation-related genes;②lipid metabolism genes;③renin angiotensin system-related genes;④nitric oxide synthase gene. However, these studies in different countries, regions or ethnic origin can get few good replication, suggesting that such research may have great regional differences. Therefore, it is necessary to verify those candidate genes in different populations.
Atherosclerosis is the important pathophysiological basis IS. Now, it has been confirmed that the inflammatory reaction was of great importance in the process of atherosclerosis. Therefore, this study based on the Han population in northern China, a region having high incidence of IS, and selected Phosphodiesterase 4D(PDE4D) and Interleukin-1(IL-1), two important components of genes regulating the inflammatory system. In this candidate genes case-control analysis, we mesured genotype and allele frequencies of four PDE4D gene's SNPs (SNP83、SNP87、SNP219、SNP220) and two IL-1 gene's SNPs (IL-1A-889、IL-1B-511) between case and control group, and evaluated the association between these two genes'SNPs and IS. Therefore, we may provide more molecular epidemiology data for comprehensively understanding the potential genetic mechanisms and more early molecular biological diagnosis indicators of IS.
Materials and methods
A case-control design was introduced. According to the different causes, the patients were divided into two subgroups:the large vessel group (thrombotic cerebral infarction group) and the small vessel group(lacunar infarction group).The subjects of large vessel group were consecutively recruited from the patients of northern China region patients with a total number of 371 cases, male 240, female 141, aged 41-74 years old, Han ethnic, no consanguinity with each other, the small vessel group were consecutively recruited from northern China region patients with a total number of 302 cases, male183,female 119, aged 41-76 years old, Han ethnic, no consanguinity with each other. The controls matched by age, sex, and ethnic orign were recruited from the same geographic region, with a total of 371, male 247, female 124. The SNPs of PDE4D SNP83、87 and IL-1A-889、IL-1B-511 were genotyped by the technique of polymerase chain reaction and restriction fragment length polymorphism(PCR-RFLP). PDE4D SNP219、220 were genotyped by means of Matrix-assisted laser desorption /ionization time-of-flight mass spectrometry primer extension (MALDI-TOF-PEX)reaction. Genotyping results were confirmed by dideoxy termination squencing method. SPSS 13.0 statistical software was used for data processing. Continuous variables were expressed as mean±SE and were assessed by One-Way ANOVA. Genotype and allele frequencies were calculated for control group and each of the patient groups. Univariate comparisons of allele and genotype distributions were analyzed by x2 test. The x2 goodness of fit test was used to test for deviation of genotype distribution from Hardy-Weinberg equilibrium. Conditional Logistic Regression was used for adjusting for IS traditional risk factors of IS. The influence of PDE4D and IL-1 gene polymorphisms on IS was indicated by odds ratio(OR) and 95% confidence interval(CI) with P<0.05 as significant criteria. Haplotype-based analysis was infered by using SHEsis online-software.
Results
1. The genotype frequencies of all SNPs conformed to the expectations of Hardy-Weinberg equilibrium(P>0.05). It was suggested that the selected samples could represent the population and were suitable for genetic analysis.
2. PDE4D gene SNPs frequencies distributions among the large vessel and control group:SNP83CC genotype and C allele frequencies of the case group were higher than the control group(CC:P=0.001; OR= 1.788; 95%CI:1.280-2.497;C:P=0.003; OR= 1.368;95%CI:1.115-1.678); SNP219 AA genotyp and A allele frequencies of the large vessels group were higher than the control group(P=0.029; OR= 1.444; 95%CI: 1.037-2.010; A:P=0.048; OR= 1.228;95%CI:1.001-1.505), and after conditional logistic regression the differece was still significant(SNP83:OR= 1.479; 95%CI: 1.024-2.135; P=0.037; SNP219:P=0.046; OR=1.567; 95%CI:1.008-2.436).
3. PDE4D gene SNPs'frequencies distributions among the small vessel and control group:Significant differences of all the frequencies were not observed between these two groups.
4.PDE4D gene haplotypes'frequencies among the large vessel and control group: The frequencies of the haplotype built by SNP83 and SNP87 were normal among the patients and the controls; Among the haplotypes built by SNP219 and SNP220, AA's frequency of the case group was higher than the control group(P=0.013;OR=1.296; 95%CI:1.056-1.591), while the frequency of GA in the case group was lower than the control group(P=0.005; OR=0.650; 95%CI:0.482-0.878).
5.PDE4D gene haplotypes'frequencies among the small vessel and control group: the frequencies of CT and TT in the control group were higher than those in the case group(CT:P=0.014;OR=0.637; 95%CI:0.444-0.913;TT:P=0.004; OR=0.662; 95%CI: 0.528-0.830),while the frequency of TC in the case group was higher than which in the control group(P=0.002;OR=1.861; 95%CI:1.391-2.490).
6.IL-1SNPs'frequencies distributions among the cases and controls:the T allele's frequency of IL-1A-889 of the case group was higher than the control group (P=0.024; OR= 1.277;95%CI:1.033-1.577), and after conditional logistic regression the difference was still significant(P=0.034; OR=1.913; 95%CI:1.621-2.375). All the frequencies of genotypes and alleles of IL-1B-511 between the two groups had no significant differences.
Conclusions
1. PDE4D SNP83 might be associated with the risk of thrombotic cerebral infarction in northern han Chinese.
2. The haplotypes built by PDE4D SNP83 and SNP87 might have no association With thrombotic cerebral infarction. The haplotype AA built by SNP219 and SNP220 might be at-risk haplotype of thrombotic cerebral infarction, meanwhile, GA might be be protective haplotype.
3.The haplotype TC built by PDE4D SNP83 and SNP87 might be at-risk haplotype of lacunar infarction.
4.The haplotype CT and TT built by PDE4D SNP83 and SNP87 might be protective haplotypes of lacunar infarction.
5.PDE4D gene might be associated with the risk of lacunar infarction in northern han Chinese.
6.Although t he result of our study supports the conclusion of deCODE, there still be some difference because of the difference of regions and ethnics.
7. IL-1A-889 T allele might be associated with thrombotic cerebral infarction in northern han Chinese, but it need to be tested by larger studies.
8. IL-1B-511 might have no association with IS in northern han Chinese.
9. The association between IL-1 polymorphism and IS might be affected by the difference of regions、ethnics and nationalities.
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9 Almeida AM, Murakami Y, Layton DM, et al. Hypomorphic promoter mutation in PIGM causes inherited glycosy phosphatidylinositol deficiency. Nat Med,2006,12:846-851.
10 McPherson R, Persemlidis A, Kavaslar N, et al. A common allele on chromosome 9 associated with coronary heart disease. Science,2007,316:1488-1491.
11 Helgadottir A, Thoreleifsson G, Manolescu A, et al. A common variant on chromosome 9p12 affects the risk of myocardial infarction. Science,2007,316:1491-1493.
12 Rioux JD, Xavier RJ, Taylor KD, et al. Genome-wide association study identifies new susceptibility loci for Crohn disease and implicates autophgyin disease pathogenesis. Nat Genet,2007,39:596-604.
13 Haiman CA, Patterson N, Freedman ML, et al. Multiple regions within 8q24 independently affect risk for prostate cancer. Nat Genet,2007,39:638-644.
14 Gudmandsson J, Sulem P, Manolescu A, et al. Genome-wide association study identifies a second prostate cancer susceptibility variant at 8q24. Nat Genet,2007,39:631-637.
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21 Eichner JE, Dunn ST, Perveen G, et al. Apolipoprotein E polymorphism and cardiovascular disease:a HuGE review. Am J Epidemiol,2002,155:487-495.
22 Rothwell N. Interleukin-1 and neuronal injury:mechanisms, modification, and therapeutic potential. Brain Behav Immun,2003,17:1522157.
23 Zee R, Cook NR, Cheng S, et al. Polymorphism in the P-selectin and interleukin-4 genes as determinants of stroke:a population-based, prospective genetic analysis. Hum Molec Genet, 2004,13:389-396.
24 Klein RL, Ascencao JL, MironovaM, et al. Effect of inflammatory cytokines on the metabo-lism of low-density lipop roteins by human vascular endothelial cells. Meta-bolism,2001,50: 99-106.
25 Bioque G, Crusius JB, Koutroubakis I, et al. Allelic polymorphism in IL-1β and IL-1 receptor antagonist (IL-1 Ra) genes in inflammatory bowel disease. Clin Exp Immunol,1995,102: 379-383.
26 Gidron Y, Gilutz H, Berger R, et al. Molecular and cellular interface between behavior and acute coronarysyndromes[J]. Cardiovasc Res,2002,56:15-21.
27 Um JY, Moon KS, Lee KM, et al. Association of interleukin-1 a gene polymorphism with cerebral infarction. Brain ResMol Brain Res,2003,5,23:41-46.
28 Helgadottir A, Manolescu A, Thorleifsson G, et al. The gene encoding 5-lipoxygenase activating protein confers risk of myocardial infarction and stroke. Nature Genet,2004, 36:233-239.
29 Slowik A, Turaj W, Dziedzic T, et al. DD genotype of ACE gene is a risk factor for intracerebral hemorrhage. Neurology,2004,63:359-361.
30 Casas JP, Hingorani AD, Bautista T, et al. Meta-analysis of studies in ischemic stroke: thirty-two genes involving approximately 18,000 cases and 58,000 controls. Arch Neurol,2004, 61:1652-1661.
31 Berger K, Stogbauer F, Stoll M, et al. The glu298asp polymorphism in the nitrc oxide synthase 3 gene is associated with the risk of ischemic stroke in two large independent case-control studies. Hum. Genet,2007,121:169-178.
32 Gretarsdottir S, Sveinbjornsdottir S, Jonsson H, et al. Localization of a susceptibility gene for common forms of stroke to 5q12. Am. J. Hum. Genet,2002,70:593-603.
33 Gretarsdottir S, Thorleifsson G, Reynisdottir ST, et al. The gene encoding phosphodiesterase 4D confers the risk of ischemic stroke. Nature Genet.2003,35:131-138. Note:Erratum: Nature Genet,37:555 only,2005.
34 Kostic MM, Erdogan S, Rena G, et al. Altered expression of PDE1 and PDE4 cyclic nucleotide phosphodiesterase isoforms in 7-oxo-prostacyclin-preconditoned rat heart.J Mol Cell Cardiol,1997,29:3135-3146.
35 Houslay MD, Adams DR. PDE4 cAMP phosphodiesterase:modular enzymes that orchestrate signalling cross-talk, desensitization and compartmentalization. Biochem J,2003,370:1-18
36杨晓军.磷酸二酯酶4D基因与缺血性卒中.国外医学脑血管疾病分册,2006,12：310-312.
37 Alberts MJ. Genetics of cerebrovascular disease. Stroke,2004,35:342-344.
38 Kostulas K, Gretarsdottir S, Kostulas V, et al. PDE4D and ALOX5AP genetic variants and risk for Ischemic Cerebrovascular Disease in Sweden. Journal of the Neurological Sciences, 2007,263:113-117.
39 Saleheen D, Bukhari S, Haider SR, et al. Association of phosphodiester-ase 4D Gene With Ischemic Stroke in a Pakistani Population. Stroke,2005,36:2270-2277.
40 Banerjee I, Gupta V, Ahmed T, et al. Inflammatory system gene polymorphism and the risk of stroke:A case-control study in an Indian population.Brain Research Bulletin 2008,75: 158-165.
41 Bevan S, Porteous L, Sitzer M. Phosphodiesterase 4D Gene, Ischemic Stroke, and Asymptomatic Carotid Atherosclerosis. Stroke,2005,36:949-953.
42 Victoria HB, Sunhee KR, Brian KR, et al .Association of Phosphodiesterase 4D Polymorphisms With Ischemic Stroke in a US Population Stratified by Hypertension Status. Stroke,2006,37: 1385-1390.
43 Woo D, Kaushal R, Kissela B. Association of Phosphodiesterase 4D With Ischemic Stroke. Stroke,2006,37:371-376.
44 Nakayama T, Asai S, Sato N, et al. Genotype and Haplotype Association Study of the STRK1 Region on 5q12 Among Japanese.A Case-Control Study. Stroke,2006,37:69-76.
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8 Gretarsdottir S, Thorleifsson G, Reynisdottir ST, et al. The gene encoding phosphodiesterase 4D confers the risk of ischemic stroke. Nature Genet,2003,35:131-138. Note:Erratum: Nature Genet,37:555 only,2005.
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11 Saleheen D, Bukhari S, Razi Haider SR, et al. Association of phosphodiesterase 4D Gene With Ischemic Stroke in a Pakistani Population. Stroke,2005,36:2270-2277.
12 Banerjee I, Gupta V, Ahmed T, et al.Inflammatory system gene polymorphism and the risk of stroke:A case-control study in an Indian population. Brain Research Bulletin,2008,75:158-165.
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1吴江.神经病学.第一版.北京：人民卫生出版社,2006：150-153.
2 Jonathan Rosand, David Altshuler, et al. Human Genome Sequence Variation and the Search for Genes Influencing Stroke. Stroke,2003,34:2512-2517.
3 Dichgans M, Hugh S, et al. Genetic Association Studies in Stroke Methodological Issues and Proposed Standard Criteria. Stroke,2005,36:2027-2031.
4金丽江.2005年卒中和脑血管疾病的遗传学研究进展.中国卒中杂志,2005,2：146-147.
5冯作化.医学分子生物学.第一版.北京：人民卫生出版社,2006：219-220.
6 Richards AJ, Laidlaw M, Meredith SP, et al. Missense and silent mutations in COL2A1 result in stickler syndrome but via different molecular mechanisms. Hum Mutat,2007,28:639.
7 Lenski C, Kooy RF, Reyniers E, et al. The reduced expression of the HADH2 protein causes X-linked mental retardation, choreoathetosis, and abnormal behavior. AM J Hum Genet,2007, 80:372-377.
8 Borck G, Zarhrate M, Cluzeau C,et al. Father-to-daughter transmission of Cornelia de lange syndrome caused by a mutation in the 5 untranslated region of the NIPBL gene. Hum Mutat, 2006,27:731-735.
9 Almeida AM, Murakami Y, Layton DM, et al. Hypomorphic promoter mutation in PIGM causes inherited glycosy phosphatidylinositol deficiency. Nat Med,2006,12:846-851.
10 McPherson R, Persemlidis A, Kavaslar N, et al. A common allele on chromosome 9 associated with coronary heart disease. Science,2007,316:1488-1491.
11 Helgadottir A, Thoreleifsson G, Manolescu A, et al. A common variant on chromosome 9p12 affects the risk of myocardial infarction. Science,2007,316:1491-1493.
12 Rioux JD, Xavier RJ, Taylor KD, et al. Genome-wide association study identifies new susceptibility loci for Crohn disease and implicates autophgyin disease pathogenesis. Nat Genet,2007,39:596-604.
13 Haiman CA, Patterson N, Freedman ML, et al. Multiple regions within 8q24 independently affect risk for prostate cancer. Nat Genet,2007,39:638-644.
14 Gudmandsson J, Sulem P, Manolescu A, et al. Genome-wide association study identifies a second prostate cancer susceptibility variant at 8q24. Nat Genet,2007,39:631-637.
15 Yeager M, Orr N, Hayes RB, et al. Genome-wide association study of prostate cancer identifies a second risk locus at 8q24. Nat Genet,2007,39:645-649.
16 Kryukov GV, Pennacchio LA, Sunyaev SR. Most rare missense alleles are deleterious in humans:implications for complex disease and association studies. Am J Hum Genet,2007,80: 727-739.
17 Kannel WB, Wolf PA, Verter J. et al.(1970) Epidemiologic assessment of the role of blood pressure in stroke. The Framingham study. JAMA,1970,214:301-310.
18 Kiely DK, Wolf PA, Cupples LA, et al. Familial aggregation of stroke. The Framingham Study. Stroke,1993,24:1366-1371.
19 Hrubec Z, Robinette CD. The study of human twins in medical research. New Engl J Med, 1984,310:435-441.
20 Carmelli D, DeCarli C, Swan GE, et al. Evidence for genetic variance in white matter hyperintensity volume in normal elderly male twins. Stroke,1998,29:1177-1181.
21 Eichner JE, Dunn ST, Perveen G, et al. Apolipoprotein E polymorphism and cardiovascular disease:a HuGE review. Am J Epidemiol,2002,155:487-495.
22 Rothwell N. Interleukin-1 and neuronal injury:mechanisms, modification, and therapeutic potential. Brain Behav Immun,2003,17:1522157.
23 Zee R, Cook NR, Cheng S, et al. Polymorphism in the P-selectin and interleukin-4 genes as determinants of stroke:a population-based, prospective genetic analysis. Hum Molec Genet, 2004,13:389-396.
24 Klein RL, Ascencao JL, MironovaM, et al. Effect of inflammatory cytokines on the metabo-lism of low-density lipop roteins by human vascular endothelial cells. Meta-bolism,2001,50: 99-106.
25 Bioque G, Crusius JB, Koutroubakis I, et al. Allelic polymorphism in IL-1β and IL-1 receptor antagonist (IL-1 Ra) genes in inflammatory bowel disease. Clin Exp Immunol,1995,102: 379-383.
26 Gidron Y, Gilutz H, Berger R, et al. Molecular and cellular interface between behavior and acute coronarysyndromes[J]. Cardiovasc Res,2002,56:15-21.
27 Um JY, Moon KS, Lee KM, et al. Association of interleukin-1 a gene polymorphism with cerebral infarction. Brain ResMol Brain Res,2003,5,23:41-46.
28 Helgadottir A, Manolescu A, Thorleifsson G, et al. The gene encoding 5-lipoxygenase activating protein confers risk of myocardial infarction and stroke. Nature Genet,2004, 36:233-239.
29 Slowik A, Turaj W, Dziedzic T, et al. DD genotype of ACE gene is a risk factor for intracerebral hemorrhage. Neurology,2004,63:359-361.
30 Casas JP, Hingorani AD, Bautista T, et al. Meta-analysis of studies in ischemic stroke: thirty-two genes involving approximately 18,000 cases and 58,000 controls. Arch Neurol,2004, 61:1652-1661.
31 Berger K, Stogbauer F, Stoll M, et al. The glu298asp polymorphism in the nitrc oxide synthase 3 gene is associated with the risk of ischemic stroke in two large independent case-control studies. Hum. Genet,2007,121:169-178.
32 Gretarsdottir S, Sveinbjornsdottir S, Jonsson H, et al. Localization of a susceptibility gene for common forms of stroke to 5q12. Am. J. Hum. Genet,2002,70:593-603.
33 Gretarsdottir S, Thorleifsson G, Reynisdottir ST, et al. The gene encoding phosphodiesterase 4D confers the risk of ischemic stroke. Nature Genet.2003,35:131-138. Note:Erratum: Nature Genet,37:555 only,2005.
34 Kostic MM, Erdogan S, Rena G, et al. Altered expression of PDE1 and PDE4 cyclic nucleotide phosphodiesterase isoforms in 7-oxo-prostacyclin-preconditoned rat heart.J Mol Cell Cardiol,1997,29:3135-3146.
35 Houslay MD, Adams DR. PDE4 cAMP phosphodiesterase:modular enzymes that orchestrate signalling cross-talk, desensitization and compartmentalization. Biochem J,2003,370:1-18
36杨晓军.磷酸二酯酶4D基因与缺血性卒中.国外医学脑血管疾病分册,2006,12：310-312.
37 Alberts MJ. Genetics of cerebrovascular disease. Stroke,2004,35:342-344.
38 Kostulas K, Gretarsdottir S, Kostulas V, et al. PDE4D and ALOX5AP genetic variants and risk for Ischemic Cerebrovascular Disease in Sweden. Journal of the Neurological Sciences, 2007,263:113-117.
39 Saleheen D, Bukhari S, Haider SR, et al. Association of phosphodiester-ase 4D Gene With Ischemic Stroke in a Pakistani Population. Stroke,2005,36:2270-2277.
40 Banerjee I, Gupta V, Ahmed T, et al. Inflammatory system gene polymorphism and the risk of stroke:A case-control study in an Indian population.Brain Research Bulletin 2008,75: 158-165.
41 Bevan S, Porteous L, Sitzer M. Phosphodiesterase 4D Gene, Ischemic Stroke, and Asymptomatic Carotid Atherosclerosis. Stroke,2005,36:949-953.
42 Victoria HB, Sunhee KR, Brian KR, et al .Association of Phosphodiesterase 4D Polymorphisms With Ischemic Stroke in a US Population Stratified by Hypertension Status. Stroke,2006,37: 1385-1390.
43 Woo D, Kaushal R, Kissela B. Association of Phosphodiesterase 4D With Ischemic Stroke. Stroke,2006,37:371-376.
44 Nakayama T, Asai S, Sato N, et al. Genotype and Haplotype Association Study of the STRK1 Region on 5q12 Among Japanese.A Case-Control Study. Stroke,2006,37:69-76.