Ⅱb型高脂蛋白血症低密度脂蛋白受体相关蛋白四核苷酸重复序列(TTTC)n基因多态性研究

目的　探索中国人Ⅱb型高脂蛋白血症患者低密度脂蛋白受体相关蛋白 (LRP)四核苷酸重复序列 (TTTC)n基因多态性与血清脂质、脂蛋白和载脂蛋白水平的关系。方法　采用聚合酶链反应结合聚丙烯酰胺凝胶电泳和银染技术 ,分别对 72例Ⅱb型高脂蛋白血症患者及 10 1例正常健康人的LRP(TTTC)n基因多态性进行基因型检测 ,同时对TG、TC、LDL C、HDL C、ApoA、ApoB浓度进行测定。结果　Ⅱb型高脂蛋白血症患者的TG、TC、LDL C、ApoB较正常对照组显著性升高 (P <0 .0 1) ;而且Ⅱb型高脂蛋白血症组TC、LDL C、ApoB由 5 / 5、5 / 6、向 6 / 6基因型变化中存在渐降趋势 ,且有统计学意义 (P <0 .0 1) ,而正常对照组也有类似的变化趋势 ,但无统计学意义 (P >0 .0 5 )。结论　Ⅱb型高脂蛋白血症患者LRP四核苷酸重复序列 (TTTC)n不同基因型与血脂水平有关     

Fluorescence-based, nonradioactive method for efficient detection of the pentanucleotide repeat (TTTTA)(n) polymorphism in the apolipoprotein(a) gene

BACKGROUND: The apolipoprotein(a) [apo(a)] gene is a major predictor of plasma lipoprotein(a) concentrations, an independent risk factor for cardiovascular disease. The apo(a) gene contains a pentanucleotide repeat (PNR) polymorphism, 1.4 kb upstream from the apo(a) gene reading frame. This polymorphism has been suggested to be important in control of apo(a) gene expression. METHODS: We developed a fluorescence-based, nonradioactive procedure to detect the PNR polymorphism. After amplification of the polymorphism by PCR, the respective PCR products were separated by denaturing polyacrylamide gel electrophoresis and detected using a 3'-end fluorescently labeled oligonucleotide as a probe. We used the method to characterize the PNR polymorphism pattern in 313 individuals, 195 Caucasians and 118 African Americans. The new method efficiently separated DNAs corresponding to the different PNR repeats. RESULTS: Among both ethnic groups, alleles containing eight PNRs were most common. Smaller PNRs were more common among African Americans, and larger PNRs were more common among Caucasians. CONCLUSIONS: We developed a nonradioactive technique that separates the PNR polymorphism in the apo(a) gene and can be used in other studies involving closely sized polymorphisms.     

The association of serum lipoprotein(a) levels, apolipoprotein(a) size and (TTTTA)(n) polymorphism with coronary heart disease.

BACKGROUND: The association between lipoprotein(a) levels, apolipoprotein(a) size and the (TTTTA)(n) polymorphism which is located in the 5' non-coding region of the apo(a) gene was studied in 263 patients with severe coronary heart disease and 97 healthy subjects. METHODS: Lp(a) levels were measured by ELISA, apo(a) isoform size was determined by SDS-agarose gel electrophoresis, and analysis of the (TTTTA)(n) was carried out by PCR. For statistical calculation, both groups were divided into low (at least one apo(a) isoform with < or = 22 Kringle IV) and high (both isoforms with >22 KIV) apo(a) isoform sizes, and into low number (<10 in both alleles) and high number of (> or =10 at least one allele) TTTTA repeats. RESULTS: Lp(a) levels were higher (P=0.007), apo(a) isoforms size < or =22 KIV and TTTTA repeats > or = 10 were more frequent (P=0.007 and 0.01) in cases than in controls. Lp(a) levels were found to be increased with low apo(a) weight in both groups (both P<0.0001). In multivariate logistic regression analysis, only the Lp(a) levels (P=0.005) and (TTTTA)(n) polymorphism (P=0.002) were found to be significantly associated with CHD. CONCLUSION: Nevertheless, these results indicate that in CHD patients the (TTTTA)(n) polymorphism has an effect on Lp(a) levels which is independent of the apo(a) size.     

载脂蛋白CⅡ微卫星DNA检测的方法学建立

目的 建立准确检测载脂蛋白CⅡ（apoⅡ)微卫星DNA（TG）n(AG)m的方法。方法 采用套式聚合酶链反应扩增样品DNA，将扩增产物上样于变性聚丙烯酰胺凝胶，高压电泳，凝胶银染后按等位基因 标准判定样品基因型；对等位基因（TG）20（AG）8进行测序。结果 (TG)n(AG)m位点，共检出12个等位基因（17、18、26－35），36个基因型。（AG）m位点，共检出4个等位基因（6－9），7个基因型。DNA测序确证该等位基因为（TG）20（AG）8。结论 该法准确、快速、简便，适用于科研及大规模人群检查。     

帕金森病发病风险与单胺氧化酶B基因微卫星多态性的相关研究

目的:探讨单胺氧化酶B(monoamineoxidaseB,MAOB)基因内含子2区(GT)n微卫星多态与散发性帕金森病发病风险间的关系。方法:采用扩增片段长度多态法(Amp-FLP)和微卫星荧光标记-半自动基因分型技术,对142例符合诊断标准的中国帕金森病患者和210例健康正常人,进行MAOB基因(GT)n微卫星多态分型,并通过相对风险率(relativerate,RR)与帕金森病发病风险作相关分析。结果:①病例-对照组间各等位基因频率分布差异有显著性意义(χ2=25.11,df=15,P=0.048)。其中168,170,184,186bp等位基因与帕金森病正相关,而172bp和182bp等位基因与帕金森病发病风险负相关。②按性别分层后,男性帕金森病患者中仅184bp等位基因频率明显增加(χ2=17.54,P=0.00003),182bp等位基因频率明显减少(χ2=9.10,P=0.026)。女性帕金森病患者中168,170,186,188bp等位基因频率明显增加(168bpχ2=9.55,P=0.002;170bpχ2=5.30,P=0.021;184bpχ2=24.76,P=0.000001;186bpχ2=4.35,P=0.037)。结论:MAOB基因内含子2中(GT)n多态与散发性帕金森病的发病风险有关。这种关联关系有性别差异。     

雄激素受体基因（CAG）n多态性与男性2型糖尿病血管并发症相关性

目的研究雄激素受体（AR）基因（CAG）n多态性分布频率及其与2型糖尿病血管病变的关系。方法DNA测序技术检测48例男性2型糖尿病血管并发症患者雄激素受体基因CAG串联重复序列（CAG—STR）长度,并分别与60例无血管并发症2型糖尿病患者和46例正常对照组比较。结果CAG重复次数介于13～36,平均22．2,2型糖尿病伴血管性病变组CAG重复次数23．4±3．85,无血管并发症组22．3±3．57,正常对照组21．9±3．45,糖尿病血管病变组CAG—STR长度明显高于正常对照组和无血管病变组（P〈0．05）,CAG重复次数与HDL—C水平呈正相关。结论男性2型糖尿病血管并发症与雄激素受体基因（CAG）n多态性有关,雄激素受体CAG重复序列的长度可能是糖尿病血管病变的危险因素,越长CAG重复序列并发血管病变的可能性越大。     

心房利钠肽基因多态性与中国汉族2型糖尿病患者蛋白尿关系的研究

目的：探讨心房利钠肽（ANP）基因多态性与中国汉族2型糖尿病患者蛋白尿之间的关系。方法：应用聚合酶链反应-限制性片段长度多态性（PCR-RFLP）技术对2型糖尿病患者中无蛋白尿的患者50例、微量蛋白尿患者43例、大量蛋白尿患者40例以及正常对照组42名进行ANP基因C/T多态性研究。结果：研究对象具有限制性内切酶BatXI酶切位点的T等位基因频率显著高于白种人；对照组及糖尿病各组之间T及C等位基因和基因型频率均无统计学差异。结论：ANP基因C/T多态性与中国汉族2型糖尿病患者蛋白尿无相关性，可能不是中国汉族2型糖尿病肾病的主要遗传标志。     

湖北地区汉族人群动脉硬化性脑梗死与载脂蛋白(a)5''''调控区(TTTTA)n基因多态性的关系

背景载脂蛋白(a)具有促进动脉粥样硬化斑块形成的作用,在载脂蛋白(a)5'存在一个五核苷酸重复序列(pentanuclotide repeats,PNR),对研究遗传和预测动脉硬化性脑梗死(atherosclerosis cerebral infarction,ACI)的危险性具有一定的价值,但在不同人种中与ACI的关系尚处在探索阶段,载脂蛋白(a)PNR基因多态性在汉族人群ACI发病中的意义如何?目的研究载脂蛋白(a)PNR基因多态性与ACI的关系.设计以湖北地区汉族ACI患者和正常人群为研究对象的病例-对照研究.单位一所大学医院的检验科.对象选择1998-02/1999-03武汉大学中南医院和武汉大学亚太医院神经内科确诊的ACI患者82例(ACI组)及健康体检者153例(对照组),均为无血缘关系的湖北地区汉族人.方法对所有受试者分别检测血清脂蛋白(a)、总胆固醇、高密度脂蛋白胆固醇(high density lipoprotein cholesterol,HDL-C)、低密度脂蛋白胆固醇(ligh density lipoprotein cholesterol,LDL-C)、三酰甘油、载脂蛋白AⅠ及载脂蛋白B水平.同时采用聚合酶链反应结合高压聚丙烯酰胺凝胶电泳检测载脂蛋白(a)5'调控区五核苷酸重复序列基因多态性,并加以对照分析.结果ACI患者脂蛋白(a)[(239.9±225 4)mg/L]、总胆固醇[(4.76±0.74)mmol/L]、三酰甘油[(1.74±0.60)mmol/LI、LDL-C[(2.84±0.63)mmol/L]水平明显高于对照组[(133.5±97.7)mg/L,(4.29±0.72)mmol/L,(1.05±0.52)mmol/L,(2.84±0.63)mmol/L],HDL-C水平[(0.88±0.17)mmol/L]明显低于对照组[(1.03±0.35)mmol/L],差异均有显著性意义(t=3.65～9.18,P均＜0.01),载脂蛋白A Ⅰ[(1.13±0.15)和(1.25±0.19)mmol/L]、载脂蛋白B[(0.93±0.12)和(0.89±0.15)mmol/L],与对照组比较差异无显著性意义;ACI组中重复序列数为5的等位基因频率(0.098)明显高于正常对照组(0.026),差异有显著性意义(x2=5.62,P＜0.05),重复序列数为9的等位基因频率(0.073)明显低于对照组(0.213),差异有显著性意义(x2=7.83,P＜0.01),且重复序列数为5的等位基因还与血浆TC下降和Lp(a)升高有关联.结论提示Apo(a)PNR基因多态性与人群易感ACI有关,可能在一定程度上参与ACI的发生和发展过程.     

利钠肽的研究进展

利钠肽家族作为内分泌激素，能够通过调节心脏和。肾脏的功能达到维持机体的内稳态。对利钠肽的最新研究表明，心力衰竭等心脏病患者的利钠肽水平升高，升高的利钠肽提示心血管事件的高危险性，监测利钠肽能够帮助诊断和判断预后。利钠肽能够抑制心肌的过度增殖和纤维化，具有改善心肌梗死和心衰时的心肌重构作用。脑钠肽是一种最重要的利钠肽，它可以作为心衰和其他心血管疾病的重要生物标记物，能够帮助我们了解肺动脉高压和粥样硬化性血管病等心血管疾病的进展。另外，合成的多种利钠肽如nesiritide已经被试验用来治疗急性充血性心衰。针对多种重组利钠肽的多个临床研究还在实验阶段，主要的方向包括它们在心脏手术中对心脏、肾脏的保护作用和抑制心肌重构的作用。     

Association of the insertion/deletion gene polymorphism of the apolipoprotein B signal peptide with myocardial infarction in Tunisian patients

BACKGROUND: Numerous polymorphisms of the apolipoprotein B (APOB) gene have been described. Particularly, the insertion/deletion (Ins/Del) polymorphism located in the coding part of the signal peptide of apoB, associated with modification of lipid concentrations and the risk of coronary artery disease and/or myocardial infarction (MI), has been reported in the general population. Moreover, conflicting results emerge from the literature and suggest that the effect is context-dependent. In the present study, the first investigation of the Ins/Del polymorphism of the APOB gene in Tunisian patients with MI, we examined a possible association between this polymorphism and MI in a subgroup of the Tunisian population. METHODS: A total of 318 Tunisian patients with MI and 368 healthy controls were included in the study. Genomic DNA was extracted from white blood cells, and the Ins/Del polymorphism was determined by electrophoresis in polyacrylamide gels after PCR amplification. A binary logistic regression analysis was performed to test how the association between MI and Ins/Del polymorphism is independent from confounding factors. RESULTS: A significant difference in genotype distribution and allele frequency was observed between patients and controls. Patients with MI had a frequency of 7.2% for the Del/Del genotype, 39.6% for the Ins/Del genotype, and 53.1% for the Ins/Ins genotype. Controls had a frequency of 3.0% for the Del/Del, 32.1% for the Ins/Del and 64.9% for the Ins/Ins genotype (chi2=12.93, p=0.002). The MI patient group showed a significantly higher frequency of the Del allele compared to controls (27.1% vs. 19.1%; chi2=12.50, p=0.0004). In comparison to the Ins/Ins homozygotes, the odds ratio (95% confidence interval) for MI was 1.51 (1.09-2.07) for Ins/Del heterozygotes and 2.95 (1.40-6.22) for Del/Del homozygotes. In multivariate analysis, age (p=0.001), smoking (p<0.001), hypertension (p=0.001), diabetes mellitus (p<0.001), and dyslipidemia (p=0.01) were independent correlates of the presence of MI, whereas the Ins/Del polymorphism (p=0.330) was not an independent predictor of MI. CONCLUSIONS: The present study shows a significant but not independent association between the Ins/Del polymorphism of the APOB gene and MI in the Tunisian population.     

应用实时荧光定量PCR技术分析UGT1A1基因启动子区A(TA)_nTAA多态性

目的建立实时荧光定量聚合酶链反应(PCR)技术检测尿苷二磷酸葡糖醛酸基转移酶1A1(UGT1A1)基因启动子区A(TA)_nTAA序列多态性的方法。方法以16例Gilbert综合征患者和66例健康对照个体为研究对象,抽提基因组DNA,通过DNA测序确定UGT1A1基因启动子区A(TA)_nTAA序列多态性。同时,设计1对引物和2条TaqMan MGB探针,2条探针的5′端分别标记FAM和VIC染料,探针的3′端则均以MGB修饰。使用实时荧光定量PCR方法扩增并检测研究对象的UGT1A1基因启动子区A(TA)_nTAA多态性序列,与测序法比较,验证实时荧光定量PCR方法的灵敏度和特异度。结果应用荧光定量PCR技术检测,16例Gilbert综合征患者的UGT1A1基因启动子区A(TA)_nTAA多态性序列均为A(TA)7TAA,46例健康对照个体A(TA)_nTAA多态性序列为A(TA)6TAA,其余20例健康对照个体A(TA)_nTAA多态性序列为A(TA)6TAA/A(TA)7TAA杂合多态性。上述结果与DNA测序结果完全一致。结论通过实时荧光定量PCR技术检测UGT1A1基因启动子区A(TA)_nTAA序列多态性的方法具有灵敏度高、特异度强及操作简单等特点,可在临床推广应用。     

儿茶酚胺氧位甲基转移酶基因多态性与抑郁症的关联

目的:探讨中国汉族人群抑郁症患者与儿茶酚胺氧位甲基转移酶(COMT)基因多态性之间的关系.方法:抽取110例抑郁症患者(抑郁症组)作研究,以114例正常人(正常对照组)作对照.应用聚合酶链式反应(PCR)扩增技术测定所有研究对象的COMT基因的基因型和等位基因.结果:COMT基因的3种基因型(A/A,A/G和G/G)在抑郁症组的分布分别为2.7%、25.5%和71.8%,在正常对照组分别为5.3%、49.1%和45.6%,两组间差异有显著性(P＜0.05).结论:COMT基因的多态性与抑郁症之间有显著关联.     

Reference intervals and decision limits for B-type natriuretic peptide (BNP) and its precursor (Nt-proBNP) in the elderly

BACKGROUND: Elderly patients have the highest prevalence of heart failure (HF). The aims of the study were to establish a reference interval for B-type natriuretic peptide (BNP) and (Nt-proBNP) in elderly people, and to identify clinically relevant decision limits based on long-term outcome. METHODS: Plasma concentrations of BNP and Nt-proBNP were measured from two elderly populations: 218 healthy subjects (mean age 73 years, population I), and 474 patients (mean age 73 years, population II) with symptoms associated with HF. Study population II was followed for 6 years with registration of all cardiovascular mortality. RESULTS: An association between both BNP and Nt-proBNP concentrations and age was found. The upper limit for the reference intervals in the healthy elderly (population I) was: BNP< or =28 pmol/L (< or =97 ng/L), and Nt-proBNP< or =64 pmol/L (< or =540 ng/L). Based on cardiovascular mortality, decision limits for BNP (approximately 50 pmol/L, approximately 170 ng/L) and Nt-proBNP ( approximately 200 pmol/L, approximately 1700 ng/L) (population II) were determined. CONCLUSIONS: Besides establishing reference intervals for BNP and Nt-proBNP in an elderly population, a higher clinically relevant decision limit for BNP and Nt-proBNP was identified, indicating additive prognostic information of the peptides on top of measurements by echocardiography. Therefore, both reference intervals and decision limits should be included in clinical practice.     

基因学干预研究:血红素氧合酶1微卫星多态性表达水平上调的作用

背景人类血红素氧合酶1(Heme Oxygenase-1,HO-1)基因启动子区域有一双核苷酸(GT)n重复序列,有高度多态性,又称为微卫星多态性,体外实验表明通过测定GT重复次数可间接了解人体内HO-1的表达水平.目的探讨HO-1基因启动子双核苷酸GT n重复序列的微卫星多态性与冠状动脉支架术后再狭窄的关系.设计以接受冠状动脉支架置入术的冠心病患者为研究对象的病例对照研究.单位一所大学医院的心内科病房.对象研究对象为1996-04/2002-05北京大学第一医院心内科病房成功接受冠状动脉支架置入术的冠心病患者,共118例.纳入标准支架术后3个月以上行冠状动脉造影随访的冠心病患者;排除标准冠状动脉造影显示原靶病变管腔直径狭窄＜50%和冠状动脉造影随访时间＜3个月的冠心病患者.入选患者年龄(62±10)岁,男92例,女26例,所有患者均签署知情同意书.根据美国心肺血液协会的标准定义,将患者分为支架内再狭窄组与无再狭窄组,分别为68例和50例.方法提取患者外周血DNA,经PCR扩增HO-1微卫星序列后采用Spreadex凝胶电泳来进行基因分型.主要观察指标HO-1基因启动子微卫星基因型频率及其与再狭窄的关系. 结果携带GT重复＜25次等位基因患者的再狭窄率为47.5%,携带两条GT重复均≥25次等位基因患者的再狭窄率为68.4%(P＜0.05).经多元回归分析校正冠心病危险因素及介入治疗的相关影响因素后,两组患者的再狭窄率差异仍有显著性意义(OR=0.418,95%可信区间0.197～0.887,P＜0.05).结论HO-1基因启动子微卫星多态性与再狭窄相关,对冠心病患者冠状动脉支架置入术后的二级预防有十分重要的意义.     

诱导型一氧化氮合酶基因启动子区微卫星多态与急性冠状动脉综合征

目的:研究中国汉族人群诱导型一氧化氮合酶(NOS2A)基因启动子区-2.5kb微卫星CCTTT串联重复序列多态与急性冠状动脉综合征(ACS)的相关性。方法:应用微卫星分析技术,检测CCTTT微卫星多态在117例ACS患者和125名健康对照者中的频率分布。结果:根据CCTTT微卫星多态对NOS2A基因转录水平的影响,将CCTTT串联重复序列分为S(重复次数≤13)和L(重复次数>13)2类,基因型分布在ACS组(S/S=48,S/L=64,L/L=5)和对照组(S/S=56,S/L=49,L/L=20)间存在显著差异(χ2=11.354,P=0.003)。对照组的L/L基因型频率显著高于ACS组,r为0.234(95%CI=0.085～0.647,P=0.003),可能具有保护性作用。分层研究发现,女性ACS与对照组相比,基因型分布存在显著差异(χ2=8.134,P=0.017)。结论:NOS2A基因启动子区CCTTT微卫星多态可能与中国汉族ACS,尤其与女性患者的遗传易感性有关。     

A novel class of miniature inverted repeat transposable elements (MITEs) that contain hitchhiking (GTCY)(n) microsatellites

The movement of miniature inverted repeat transposable elements (MITEs) modifies genome structure and function. We describe the microsatellite-associated interspersed nuclear element 2 (MINE-2), that integrates at consensus WTTTT target sites, creates dinucleotide TT target site duplications (TSDs), and forms predicted MITE-like secondary structures; a 5' subterminal inverted repeat (SIR; AGGGTTCCGTAG) that is partially complementary to a 5' inverted repeat (IR; ACGAAGCCCT) and 3'-SIRs (TTACGGAACCCT). A (GTCY)(n) microsatellite is hitchhiking downstream of conserved 5'MINE-2 secondary structures, causing flanking sequence similarity amongst mobile microsatellite loci. Transfection of insect cell lines indicates that MITE-like secondary structures are sufficient to mediate genome integration, and provides insight into the transposition mechanism used by MINE-2s.