摘要
第一部分冠心病传统危险因素与冠状动脉病变的相关性研究
目的
冠心病(coronary heart disease,CHD)是严重威胁人类健康的重要疾病,在欧美国家素有“第一杀手”之称。随着我国经济的高速发展和人民生活方式的变化,该病的患病率及死亡率逐年攀升,在某些地区亦已成为人口死亡的首要原因。目前公认的冠心病危险因素包括可以控制的高血压、糖尿病、脂质代谢紊乱、吸烟、肥胖以及不可控制的年龄、性别等,但它们与冠状动脉病变程度关系的研究,国内外报道不一。因此,我们对经冠状动脉造影确诊的冠心病患者的相关危险因素进行分析,探讨冠心病传统的危险因素与冠状动脉病变程度的关系。
方法
选择2007年5月～2007年10月在山东大学齐鲁医院心内科住院并经冠状动脉造影确诊的冠心病患者156例,记录患者的临床资料,包括年龄、性别、BMI等一般情况及既往冠心病、高血压、糖尿病病史、吸烟史和血脂、血压水平等项目,并计算non-HDL-C的水平;冠状动脉病变程度由病变支数和Gensini积分表示。分析各危险因素与冠状动脉病变支数和Gensini积分的关系。
结果
1.在病变支数不同的各组间,随病变支数的增加,糖尿病所占的百分比依次增加(P＜0.01),平均年龄、SBP、BMI和non-HDL-C的水平亦递增(P＜0.05)。
2.年龄、高血压、糖尿病以及SBP、BMI、non-HDL-C的水平与Gensini积分呈正相关(r_s=0.183～0.318,P=0.0001～0.0222),HDL-C与Gensini积分呈负相关(r_s=-0.169,P=0.0349)。
结论
糖尿病、年龄、SBP、BMI和non-HDL-C与冠状动脉病变支数有关。年龄、高血压、糖尿病、SBP、BMI和non-HDL-C与Gensini积分成正相关,HDL-C与Gensini积分呈负相关。
第二部分基因组扫描筛选冠心病易感基因位点的初步研究
目的
虽然多种冠心病危险因素已获得国内外的公认,但仍有许多冠心病患者不能用已知的危险因素解释。流行病学调查、家系及双生子研究结果显示冠心病有明显的遗传倾向,因此,从基因水平探讨冠心病发病的遗传易感机理成为近年研究的热点。对冠心病的基因研究目前主要基于两种策略:即候选基因的筛查与全基因组扫描。国内对冠心病的遗传学研究主要涉及前者,全基因组扫描研究尚未见报道。为了查找与验证中国冠心病群体中染色体的关联区域和易感基因,我们采用DNA混合池(DNA pooling)的方法,对冠心病患者及对照者进行基因组扫描,为发现与克隆冠心病新的致病基因打下基础。
方法
1.选择2007年5月～2007年10月在山东大学齐鲁医院心内科住院并经冠状动脉造影确诊的冠心病患者156例为患者组,对照组选择山东省血液中心的健康献血者450例,所有研究对象均来自山东地区,个体之间无血缘关系。
2.用改进的酚-氯仿法分别提取其基因组DNA,然后测量提取后的DNA浓度,分别稀释到20ng/μL,在上述稀释好的DNA样品中每份各取10uL混合在同一管内,作为一个DNA池,分别构建了患者组和对照组两个混合池。
3.以平均间隔10 cM(厘摩)的遗传距离选择覆盖2号、3号、14号、16号和X染色体基因组的98个微卫星遗传标记,逐一对患者组与对照组的DNA混合池样本进行DNA扩增,然后在毛细管遗传分析仪上进行基因组扫描与分型,用Genemapper软件收集数据。
4.分别计算每一位点各等位基因所占所有等位基因峰高的荧光强度数值的比率,用Clump软件进行关联统计分析,计算出x~2值与P值。将P＜0.05定为差异有统计学意义。
结果
1.基因组扫描与分型结果:经过反复试验,最终获得了所有98个遗传标记的基因分型结果。其中97个遗传位点呈现多态性,16号染色体上的D16S3103位点在该人群中的等位基因只有一种,在该人群中无多态性。
2.冠心病组与对照组等位基因分布频率差异比较的结果:在3号染色体上的D3S1278位点(3q13.31)x~2=23.778215,P=0.000500;D3S1300位点(3p14.2)x~2=27.640001,P=0.000300;16号染色体上的D16S520位点(16q24.1)x~2=30.611959,P=0.000700;D16S3046位点(16p12.2)x~2=14.989625,P=0.009499;X染色体上的DXS1227位点(Xq27.2)x~2=17.482637,P=0.023998;这五个遗传标记位点的等位基因频率在患者组与对照组之间的差异有统计学意义,显示与冠心病存在关联。
结论
在山东地区人群冠心病患者中,染色体3q13.31、3p14.2、16q24.1、16p12.2和Xq27.2区域存在与冠心病的关联位点,在关联区域内可能有致病因子,如易感基因或调控因子的存在,需要进一步对上述区域进行高密度遗传标记的精细定位,进而筛查冠心病的易感基因。
意义
本研究在国内首次采用基因组扫描关联分析的方法对冠心病易感基因进行初步筛选,在3号、16号和X染色体上发现了与冠心病关联的区域,为发现与克隆冠心病新的致病基因打下坚实基础。同时,我们创新性地将DNA混合池的技术引入对冠心病的基因扫描研究中,取到了理想的效果,大大减少了基因扫描的工作量,提高效率,节约试剂,为冠心病的分子遗传学研究开辟了一条新途径。
PartⅠThe analysis of risk factors for the degree of coronary artery lesions in coronary heart disease
Objective
Coronary heart disease (CHD) is the leading cause of death in developed countries and as we have approached the same life style in China, it is expected to be the major cause of morbidity and mortality nationwide. Classic epidemiologic studies have revealed many risk factors for CHD, including age, sex, hypertension, dyslipidemia, obesity, type 2 diabetes mellitus (T2DM), and tobacco consumption. However, the relationship between these risk factors and the degree of coronary artery lesions was reported inconsistently. So we studied here to investigate the relationship between the risk factors of CHD and the severity of the coronary artery lesions.
Methods
156 CHD cases which had been confirmed by coronary angiography (CAG) in Qilu Hospital from May 2007 to October 2007 were selected. Cardiovascular risk factors including age, gender, hypertension, T2DM, smoking, dyslipidemia and obesity were recorded. Non-HDL-C was calculated. The extent and severity of angiographic CHD was diagnosed by the number of diseased vessels and the Gensini scores. Subsequently, the relationship between risk factors and the degree of coronary artery lesions was analyzed.
Results
1. There were significant differences in T2DM, age, SBP, BMI, non-HDL-C between groups of different diseased vessels respectively (P<0.05).
2. Significantly positive correlation was presented between Gensini scores and age, hypertension, T2DM, SBP, BMI, non-HDL-C (r_s=0.183～0.318, P=0.0001～0.0222) and a negative correlation between Gensini scores and HDL-C (r_s=-0.169, P=0.0349).
Conclusions
Age, hypertension, T2DM, SBP, BMI, non-HDL-C were the risk factors for the degree of coronary artery lesions and HDL-C was the protecting factor.
PartⅡA genome-scan for the susceptibility gene loci to coronary heart disease
Objective
Classic epidemiologic studies have revealed many risk factors for CHD, however, some populations are more susceptible than others to CHD even when they share very similar environmental exposures. Previous epidemiological surveies, pedigree investigations and twin studies have suggested that there is a strong genetic component underlying this complex disease. Despite extensive exploration of many genes, strong evidence of a molecular genetic association with CHD remains to be obtained. Therefore, the positional cloning strategy offers an especially promising method to reveal the unknown etiology of disorders. Molecular genetics has presently reached a new era based on genome-wide association studies (GWAS), which has been widely used to identify chromosomal regions that might contain susceptibility genes for complex diseases such as CHD.
At present, the domestic research strategy for CHD in genomics lies in the candidate genes and studies in genome-wide scan has not been published. For finding out genetic loci associated with CHD, we performed a genome-wide scan in DNA pooling samples from CHD cases and normal controls in Shandong province. This is an innovation of the dissertation.
Methods
1. 156 CHD cases which had been confirmed by coronary angiography (CAG) in Qilu Hospital from May 2007 to October 2007 were selected. A total of 450 healthy blood donors from Shandong Blood Center were taken as the controls. All the non-related research objects came from Shandong province.
2. DNA was collected through improved phenol - chloroform extraction method and then samples were diluted to approximate concentrations of 20ng/μL. Two DNA pooling samples consisting of 156 CHD cases and 450 normal controls were constructed respectively, with each pool containing 10μL of diluted DNA from each individual.
3. A total of 98 microsatellite markers on chromosome 2, 3, 14, 16, X spaced at an average of 10 cM were selected and the two separated DNA pooling samples were amplificated and genotyped respectively.
4. Statistic analysis was performed by Chi-square method using the Clump software to compare the difference between the frequencies of each allele among the two pooling samples.
Results
1. Genome scan and genotype results: All of the 98 markers on chromosome 2, 3, 14, 16, X were amplified successfully, a total of 97 loci were polymorphic, however, D16S3103 on chromosome 16 was not informative as only one allele was observed in both case and control group.
2. Comparison of the allele frequency: Significant statistic differences were found at D3S1278, D3S1300, D16S520, D16S3046 and DXS1227 between allele frequency in cases and controls and the P- value were all<0.05. Therefore, these five loci were associated with CHD in the population of Shandong province.
Conclusions and significance
We report here the first genome-wide search for susceptibility genes in CHD in China. Five regions including 3q13.31、3p14.2、16q24.1、16p12.2 and Xq27.2 were associated with CHD in the cases of Shandong province and susceptibility genes around these regions need to be further studied.
引文
1.Gensini GG.A more meaningful scoring system for determining the severity of coronary heart disease[J].Am J Cardiol,1983,51(3):606.
2.Satoh H,Nishino T,Tomita K,et al.Risk factors and the incidence of coronary artery disease in young middle-aged Japanese men:results from a 10-year cohort study[J].Intern Med,2006,45(5):235-239.
3.Tanihata S,Nishigaki K,Kawasaki M,et al.Outcomes of patients with stable low-risk coronary artery disease receiving medical and PCI preceding therapies in Japan:J-SAP study1-1[J].Circ J,2006,70(4):365-369.
4.Brown BG,Bolson EL,Dodge HT.Dynamic mechanisms in human coronary stenosis[J].Circulation,1984,70(6):917-922.
5.Newman AB,Naydeck BL,Sutton TK,et al.Coronary artery calcification in older adults to age 99:Prevalence and risk factors[J].Circulation,2001,104(22):2679-2684.
6.邢燕,陈红,刘茹辉等.冠心病危险因素与冠状动脉病变程度的相关性研究[J].中国医药导刊,2004,6(3):159-161.
7.Breen JB,Sheedy FF,Schwart RS,et al.Coronary artery calcification detected with ultrafast CT as an indication of coronary artery disease[J].Radiology,1992,185(2):435-439.
8.江时森,柯天秀,张启高等.中国人不同性别冠状动脉钙化检出率的比较[J].心肺血管病杂志,2000,19(3):190-192.
9.Kasaoka S,Okuda F,Saton A,et al.Effect of coronary risk factors on coronary angiographic morphology in patients with ischemic heart disease[J].Jpn Circ J,1997,61(5):390-395.
10.da Luz PL,Coimbra SR.Wine,alcohol and atherosclerosis:clinical evidences and mechanisms[J].Braz J Med Biol Res,2004,37(9):1275-1295.
11.Krausse R,Leiendecker J,Herrmann G,et al.Chlamydia pneumoniae infection and restenosis in patients with coronary heart disease[J].Infection,2003,31(3):149-154.
12. Després JP. The insulin resistance-dyslipidemic syndrome of visceral obesity: effect on patients'risk[J]. Obes Res. 1998,6(Sup 1): 8S-17S.
13. Jarrett RJ. Risk factors of coronary heart disease in diabetes mellitus [J]. Diabetes, 1992,41(Sup2): 1-3.
14. Joint National Committee on Prevention,Detection,Evaluation, and Treatment of High Blood Pressure. Seventh report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure [J]. Hypertension,2003,42(6): 1206-1252.
15. National Heart Lung and Blood Institute. International activities report. Fiscal year,2001.
16. Poirier P, Lemieux I, Mauriege P, et al. Impact of waist circumference on the relationship between blood pressure and insulin : the Quebec Health Survey[J]. Hypertension,2005,45(3): 363-367.
17. Falcone C, Nespoli L, Geroldi D, et al. Dilent myocardial ischemia in diabetic and nondiabetic patients with coronary artery discase[J]. Int J Cardiol,2003,90(2-3): 219-227.
18. Jorge P, Giancarlo V, Steven H. Atherosclerosis in type-2 Diabetes mellitus and insulin resistance: mechanistic links and therapeutic targets[J]. Journal of Diabetes and its complications,2002,16(6): 401 -415.
19. Goraya TY, Leibson CL, Palumbo PJ, et al. Coronary atherosclerosis in diabetes mellitus: a population-based autopsy study[J]. J Am Coll Cardiol,2002,40(5): 946-953.
20. Summary of the second report of the National Cholesterol Education Program (NCEP) expert panel on detection. Evaluation and treatment of high blood cholesterol: in Adults(Adult Treatment Pane Ⅱ )[J]. JAMA, 1993,269(23): 3015-3023.
21. Cui Y, Blumenthal RS, Flaws, et al. Non-high-density lipoprotein cholesterol level as a predictor of cardiovascular disease mortality[J]. Arch Intern Med, 2001, 161(11): 1379-1380.
22. Frost PH, Havel RJ. Rationale for use of non-high-density lipoprotein cholesterol rather than low-density lipoprotein cholesterol as a tool for lipoprotein cholesterol screening and assessment of risk and therapy[J], Am J Cardiol, 1998,81(4A): 26B-31B.
23. Third Report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel Ⅲ) final report[J]. circulation,2002,106(25): 3143 -3421.
1.Silberberg JS,Wlodarczyk J,Fryer J,et al.Risk associated with various definitions of family history of coronary heart disease[J].The Newcastle Family History Study Ⅱ.Am J Epidemiol,1998,147(12):1133-1119.
2.Pohjola-Sintonen S,Rissanen A,Liskola P,et al.Family history as a risk factor of coronary heart disease in patients under 60 years of age[J].Eur Heart J,1998,19(2):235-239.
3.Marenberg ME,Risch N,Berkman LF,et al.Genetic susceptibility to death from coronary heart disease in a study of twins[J].N Engl J Med,1994,330(15):1041-1046.
4.Santos M,Pinto-Basto J,Rio ME,et al.A whole genome screen for association with multiple sclerosis in Portuguese patients[J].J Neuroimmunol,2003,143(1-2):112-115.
5.Sham PC,Curtis D.Monte Carlo tests for associations between disease and alleles at highly polymorphic loci[J].Ann Hum Genet,1995,59(1):97-105.
6.Barcellos LF,Klitz W,Field LL,et al.Association mapping of disease loci,by use of a pooled DNA genomic screen[J].Am J Hum Genet,1997,61(3):734-747.
7.Wellcome Trust Case Control Consortium.Genome-wide association study of 14,000 cases of seven common diseases and 3,000 shared controls[J].Nature,2007,447(7145):661-678.
8.Johnson MP,Griffiths LR.A genetic analysis of serotonergic biosynthetic andmetabolic enzymes in migraine using a DNA pooling approach[J].J Hum Genet,2005,50(12):607-610.
9.陈刚,温晓燕,朱海宁等.山东省潍坊东部地区精神分裂症1号染色体基因扫描研究[J]..中华医学遗传学杂志,2007,24(3):288-292.
10.朱海宁,陈刚,温晓燕等.山东省某地区精神分裂症患者6号染色体的基因组扫描研究[J].中华精神科杂志,2007,40(2):82-85.
11.陈刚,温晓燕,林汝湘等.山东半岛东部地区精神分裂症13号染色体的基因组扫描研究[J].精神医学杂志,2007,20(2):65-68.
12. Pajukanta P, Cargill M, Viitanen L, et al. Two loci on chromosomes 2 and X for premature coronary heart disease identified in early-and late-settlement populations of Finland[J]. Am J Hum Genet,2000,67(6): 1481 -1493.
13. Hauser ER, Crossman DC, Granger CB, et al. A genomewide scan for early-onset coronary artery disease in 438 families: the GENECARD Study[J]. Am J Hum Genet,2004,75(3): 436-447.
14. Shah S, Kraus W, Crossman D, et al. Serum lipids in the GENECARD study of coronary artery disease identify quantitative trait loci and phenotypic subsets on chromosomes 3q and 5q[J]. Ann Hum Genet,2006,70(6): 738-748.
15. Bowden DW, Rudock M, Ziegler J, et al. Coincident linkage of type 2 diabetes,metabolic syndrome,and measures of cardiovascular disease in a genome scan of the diabetes heart study[J]. Diabetes,2006,55(7): 1985-1994.
16. Wang L, Hauser ER, Shah SH, et al. Peakwide mapping on chromosome 3q13 identifies the kalirin gene as a novel candidate gene for coronary artery disease[J]. Am J Hum Genet,2007,80(4): 650-663.
17. Cernuda-Morollon E, Ridley AJ. Rho GTPases and leukocyte adhesion receptor expression and function in endothelial cells[J]. Circ Res,2006,98(6): 757-767.
18. Benedetta D. Chiodini, Cathryn M, et al. Meta-Analysis of 4 Coronary Heart Disease Genome-Wide Linkage Studies Confirms a Susceptibility Locus on Chromosome 3q[J]. Arterioscler Thromb Vasc Biol,2003,23(10): 1863-1868.
19. Francke S, Manraj M, Lacquemant C, et al. A genome-wide scan for coronary heart disease suggests in Indo-Mauritians a susceptibility locus on chromosome 16p13 and replicates linkage with the metabolic syndrome on 3q27[J]. Hum Mol Genet,2001,10(24): 2751-2765.
20. Broeckel U, Hengstenberg C, Mayer B, et al. A comprehensive linkage analysis for myocardial infarction and its related risk factors[J]. Nat Genet ,2002,30(2): 210-214.
21. Harrap SB, Zammit KS, Wong ZY, et al. Genome-wide linkage analysis of the acute coronary syndrome suggests a locus on chromosome 2[J]. Arterioscler Thromb Vasc Biol,2002,22(5): 874-878.
22. Mori Y, Otabe S, Dina C, et al. Genome-wide search for type 2 diabetes in Japanese affected sib-pairs confirms susceptibility genes on 3q,15q,and 20q and identifies two new candidate loci on 7p and 11p[J]. Diabetes,2002,51(4): 1247-1255.
23. Kissebah AH, Sonnenberg GE, Myklebust J, et al. Quantitative trait loci on chromosomes 3 and 17 influence phenotypes of the metabolic syndrome[J]. Proc Natl Acad Sci USA,2000,97(26): 14478-14483.
24. Rainwater DL, Almasy L, Blangero J, et al. A genome search identifies major quantitative trait loci on human chromosomes 3 and 4 that influence cholesterol concentrations in small LDL particles[J] . Arterioscler Thromb Vasc Biol, 1999,19(3): 777-783.
25. DeWan AT, Arnett DK, Atwood LD, et al. A genome scan for renal function among hypertensives: the HyperGEN study[J]. Am J Hum Genet,2001,68(1): 136-144.
26. Soro A, Pajukanta P, Lilja HE, et al. Genome scans provide evidence for low-HDL-C loci on chromosomes 8q23,16q24.1-24.2,and 20q13.11 in Finnish families[J]. Am J Hum Genet,2002,70(5): 1333-1340.
27. Yip AG, Ma Q, Wilcox M, et al. Search for genetic factors predisposing to atherogenic dyslipidemia[J]. BMC Genet,2003,4(S1): S100.
28. Pajukanta P, Allayee H, Krass KL, et al. Combined analysis of genome scans of dutch and finnish families reveals a susceptibility locus for high-density lipoprotein cholesterol on chromosome 16q[J]. .Am J Hum Genet,2003,72(4): 903-917.
29. Cederberg A, Gronning LM, Ahren B, et al. FOXC2 is a winged helix gene that counteracts obesity, hypertriglyceridemia, and diet-induced insulin resistance [J]. Cell,2001,106(5): 563-573.
30. Naka T, Narazaki M, Hirata M, et al. Structure and function of a new STAT-induced STAT inhibitor [J]. Nature, 1997,387(6636): 924-929.
31. Iwai N, Inagami T. Isolation of preferentially expressed genes in the kidneys of hypertensive rats[J]. Hypertension, 1991,17(2): 161 -169.
32. Schild L, Lu Y, Gautschi I, et al. Identification of a PY motif in the epithelial Na channel subunits as a target sequence for mutations causing channel activation found in Liddle syndrome[J]. EMBO J,1996,15(10): 2381-2387.
33. Wong ZY, Stebbing M, Ellis JA, et al. Genetic linkage of beta and gamma subunits of epithelial sodium channel to systolic blood pressure [J]. Lancet, 1999, 353(9160): 1222-1225.