聚合酶链反应限制性片段长度多态性检测雌激素受体基因型

目的：建立一种简便，实用，准确的人雌激素受体（ER）基因型的检测方法以利于了解基因对骨量变异的调节，方法：采用聚合酶链反应限制性片段长度多态性（PCR－RFLP）来扩增ER基因序列，用限制性内切酶PvuII对223例健康妇女和49例男性的ER基因扩增产物酶切，测定ER基因限制性片段长度多态性（RFLP），结果：汉族妇女ER基因pp,Pp,PP基因型频率分别为0．33，0．47和0．202，男性分别为0．429，0．469和0．102，结论：该方法简便，准确，重复性好，适用于常规实验，ER基因（PvuII)多态性分布有一定的种族差异性。     

聚合酶链反应限制性片段长度多太民性检测载脂蛋白E基因型

建立一种简便、准确、实用的人载脂蛋白Ｅ基因型的检测方法应用聚合酶链反应特异性扩增ａｐｏＥ基因含编码１１２位和１５８位氨基酸的基因序，扩增产物用限制性内切酶ＨｈａⅠ酶切，聚丙烯酰胺凝胶电泳后，观察酶切位点的限制性片段长度多态性图谱。结论该方法简单、快速、准确，对人体无害，适合于一般实验室开展及大规模人群调查。     

雌激素受体α基因多态性与妊娠期肝内胆汁瘀积症的相关性研究

目的探讨雌激素受体α(ERα)基因PvuII和XbaI位点的单核苷酸多态性与妊娠期肝内胆汁瘀积症(ICP)发病风险的关系.方法采用聚合酶链反应-限制性片段长度多态性(PCR-RFLP)技术对93例妊娠期肝内胆汁瘀积症患者及96例年龄与孕周皆相匹配的正常妊娠对照者进行等位基因和基因型检测.结果 ERα基因PvuII位点PP、Pp、pp基因型频率分布在ICP组分别为0.14、0.56、0.30,在对照组频率分布分别为0.17、0.48、0.35,两组间比较,差异无显著性(P>0.05).ERα基因XbaI位点XX、Xx、xx基因型频率分布在ICP组分别为0.03、0.31、0.66,在对照组频率分布分别为0.07、0.38、0.55,两组间比较,差异无统计学意义(P>0.05).ERα基因PvuII和XbaI位点等位基因频率分布在两组间差异亦无统计学意义(P>0.05).结论 ERα基因PvuII和XbaI位点的单核苷酸多态性与妊娠期肝内胆汁瘀积症的遗传易感性无关,突变基因并未增加妊娠期肝内胆汁瘀积症的发病风险.     

雌激素受体β基因多态性与广西籍汉族女性系统性红斑狼疮关联性的研究

目的探讨雌激素受体β(ERβ)基因限制性内切酶RsaⅠ酶和AluⅠ酶切多态性在广西籍汉族女性系统性红斑狼疮(SLE)患者中的分布特点。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78例女性SLE患者及100例女性正常对照者的ERβ基因AluⅠ、RsaⅠ位点进行分型,分析其基因分布情况以及临床表现与基因多态性之间的关系。结果ERβ基因型分布及等位基因频率在广西籍汉族女性SLE患者和正常对照者之间比较差异均无统计学意义(P>0.05)。RsaⅠ位点的RR型SLE患者出现狼疮性肾炎(LN)的频率以及AluⅠ位点的Aa型SLE患者出现颊部红斑的频率较其他基因型患者明显增高,差异有统计学意义(P<0.05)。结论ERβ基因RsaⅠ酶和AluⅠ酶切多态性在广西籍汉族女性SLE患者和正常人中的分布差异无统计学意义;RR基因型可能与广西籍汉族女性LN的发生相关,Aa基因型可能与患者出现颊部红斑有关。     

人β2—肾上腺素能受体基因第64位密码子突变频率的检测

建立一种简便，准确，实用的人β３－肾上腺素能受体基因第６４位密码子突变的检测方法。方法应用聚合酶链反应特异性扩增β３－肾上腺素能受体基因含编码６４位氨基酸的基因 列，扩增产物用限制性内切酶ＢｓｔＯＩ酶切，聚丙烯酰胺凝胶电泳后，观察酶切位点的限制性片段长度多态性图谱。     

低密度脂蛋白受体基因多态性与脑梗死相关性的研究

目的　探讨低密度脂蛋白受体 (LDLR)基因内含子 15PvuII多态性与动脉硬化性脑梗死的相关性 ,并对其与血脂之间的关系进行分析。方法　采用聚合链反应 (PCR)和限制性片段长度多态性 (RFLP)方法 ,检测了 5 2例脑梗死患者与 6 7例健康对照组LDLR基因含有PvuII酶切位点的等位基因频率和基因型分布。结果　脑梗死患者含有PvuII酶切位点的基因频率与正常人比较差异不显著 (P >0 0 5 )。结论　脑梗死患者血浆LDL、TC、TG水平增高 ,HDL水平降低是脑梗死的危险因素 ;LDLR基因内含子 15PvuII位点不能确定为脑梗死的遗传标记。     

HLA-DPB1等位基因与四川汉族人哮喘的相关性研究

目的:探讨HLA-DPB1等位基因与哮喘之间是否存在相关性,从基因水平探讨支气管哮喘的发病机制。方法:用聚合酶链反应-限制性片段长度多态性(polymerase chain reaction-restriction fragment length polymorphism,PCR-RFLP)分析法,检测四川汉族人HLA-DPB1基因多态性。用PCR技术对HLA-DPB1基因第2外显子(exon 2)进行体外扩增;然后用Ban II、Fok I、Dde I、Rsa I、EcoN I、BstU I 6种限制性内切酶对扩增产物进行酶切,电泳分离酶切片段,根据片段格局确定相应基因型别。结果:PCR扩增在327 bp有HLA-DPB1 exon 2目的基因产生。限制性内切酶酶切HLA-DPB1后,共检测到12种等位基因。HLA-DPB1*0201在哮喘患者组的出现频率显著低于对照组(P<0．05,RR=0．115<1)。结论:HLA-DPB1* 0201可能为支气管哮喘的抗性基因。     

雌激素受体α及β基因多态性与子宫内膜异位症易感性关系的研究

目的:探讨雌激素受体α(estrogin receptorα,ERα)及ERβ基因多态性与子宫内膜异位症易感性间的关系。方法:应用聚合酶链反应-限制性片段长度多态性(PCR-RFLP)分析检测58例子宫内膜异位症(EM)患者和107例非EM患者的ERα和ERβ基因型多态性。结果:ERα内切酶PvuⅡ位点的pp、Pp、PP基因型频率在EM组和对照组分别为34.5%和30.8%、48.3%和58.9%、15.5%和9.3%;内切酶XbaⅠ位点的xx、Xx、XX基因型频率在子宫内膜异位症组和对照组分别为56.9%和59.8%、32.8%和37.4%、8.6%和1.9%,2组比较基因分布频率差异无统计学意义(P>0.05)。ERβ内切酶AluⅠ位点的aa、Aa、AA基因型频率在EM组和对照组分别为0%和0%、19.0%和25.2%、81.0%和74.8%,内切酶RsaⅠ位点基因型频率rr、Rr、RR在EM组和对照组分别为20.7%和11.2%、32.8%和42.1%、46.6%和46.7%,2组比较基因分布频率差异无统计学意义(P>0.05)。结论:ERα及ERβ基因多态性与EM的发病易感性间无明显相关性。     

ER基因多态性与川南高氟地区人群膝骨性关节炎相关性的病例对照研究

目的：探讨川南高氟地区人群雌激素受体基因PvuⅡ和XbaⅠ核酸限制性内切酶多态性与膝骨性关节炎的相关性。方法：对川南高氟地区41例膝骨性关节炎患者及40例对照组,用聚合酶链反应限制性片段长度多态性（PCR-RFLP）的方法鉴定雌激素受体的基因型,分析雌激素受体基因多态性与膝骨性关节炎的关系及各基因型在病例组与对照组的分布。结果：41例病例组与40例对照组X基因型及P基因型频率分布差异无显著性（P〉0.05）。结论：川南高氟地区人群ER基因多态性与OA无明确相关性。     

女性SLE患者的雌激素受体α基因多态性研究

目的 分析雌激素受体α(ERα)基因XbaⅠ和PvuⅡ酶切多态性对系统性红斑狼疮(SLE)的遗传影响.方法 采用聚合酶链反应-限制性片段长度多态性(PCR-RFLP)技术对70例女性SLE患者和200名正常女性对照者的ERα基因(XbaⅠ,PvuⅡ位点)进行分型,并计算基因分布频率.结果 女性SLE患者的ERα基因多态性分布中X和P的基因频率与正常对照组并无显著性差异,ERα基因Pp基因亚型的阳性率高于正常对照组(P﹤0.05).SLE患者的ppXx基因型的阳性率明显高于正常对照组,而ppxx基因型的阳性率明显低于正常对照组(P﹤0.05).结论 女性SLE患者ERα基因的X和P的基因频率表达并无明显增高,但ERα基因酶切多态性分析中,Pp及ppXx基因型可能与女性SLE的易感性有关.     

Relation of estrogen receptor-α gene polymorphism and hormone replacement therapy to fall risk and muscle strength in early postmenopausal women

BACKGROUND. Several factors may increase fracture risk, among them reduced bone mineral density (BMD), increased bone resorption, microarchitectural deterioration of bone, increased fall risk, and decreased muscle strength. We have previously reported that PvuII polymorphism of the estrogen receptor-α (ER α) gene is associated with bone loss rate, fracture risk, and response to hormone replacement therapy (HRT) in early postmenopausal Finnish women. METHOD. We studied the influence of the ERα genotype on fall risk and muscle strength in a 5-year randomized HRT trial of 331 early postmenopausal women (subgroup of the population-based OSTPRE study, Kuopio, Finland). A 5-year postal inquiry in May 1994 included questions on falls during the previous 12 months. Grip strength was measured with dynamometer. The ERα gene polymorphism was analysed using PCR and PvuII restriction enzyme digestion. RESULTS. In all, 97 out of the 331 women reported falls. Half of those (56%) were slip falls, mostly during the winter season. In the HRT group, the ERα genotype was associated with fall risk (P = 0.002, logistic regression). The risk of falls (RR) was higher in women with the PP genotype than in those with the Pp (RR = 5.26, 95% CI 1.98-13.94, P = 0.001) or the pp (RR = 3.84, 95% CI 1.46-10.12, P = 0.007) genotype. When the falls were divided into slip (environment-related) and non-slip (endogenous) falls, the non-slip falls were associated with the genotype (P = 0.004), but the slip falls were not so clearly (P = 0.061). When all falls and non-slip falls were adjusted to the number of chronic health disorders and the variable time-since-menopause, the difference between the genotypes persisted (P = 0.003 and P = 0.010, respectively). In the non-HRT group, the ERα genotype was not associated with fall risk. The baseline or the 5-year grip strength values were not influenced by the ERα genotype. In conclusion, ER α polymorphism is associated with fall risk, especially with non-slip falls, in early postmenopausal Finnish women during the HRT. We have previously reported that, during HRT, women with the P allele have decreased fracture risk and that they may preferentially derive benefit from the positive effect of HRT on BMD. This suggests that the influence of ERα polymorphism may depend on the target tissue (bone versus the nervous system). CONCLUSIONS. In these early postmenopausal, non-osteoporotic and relatively healthy women, the increased fall risk associated with the PP genotype was not associated with increased fracture risk, possibly due to improved bone strength during the HRT although falls generally predispose to fractures.     

雌激素受体基因多态性与2型糖尿病患者骨密度的关系

目的探讨雌激素受体(ER)基因多态性与2型糖尿病患者骨密度(BMD)的相关性。方法运用双能量X线吸收法(DEXA)测定61例2型糖尿病患者的BMD,PCR-RFLP检测ER基因多态性。结果2型糖尿病组PP型的BMD与Pp型相比,在L2,L3,L4及L2～4有显著的统计学差异(P值分别为0.045,0.021,0.019和0.019);与pp型相比,在L4有显著的统计学差异(P=0.037)。Pp型与pp型相比,各部位BMD均无显著的统计学差异。ER基因型分布频率为PP型0.128,pp型0.333,Pp型0.539,PP型腰椎BMD明显低于Pp型与pp型。结论2型糖尿病易合并骨量减少和骨质疏松,ER基因PP型与2型糖尿病患者腰椎低BMD密切相关。     

Oestrogen receptor-alpha polymorphism and risk of fracture: a meta-analysis of 13 studies including 1279 cases and 6069 controls

A meta-analysis was performed to evaluate the effect of oestrogen receptor-alpha (ESR1) gene PvuII polymorphism on fracture risk. It included published data from relevant studies (up to May 2010) identified from Medline, Embase and Current Contents. The 13 included studies contained 1279 fracture cases and 6069 controls. The combined results based on these studies showed no relationship between ESR1 gene PvuII polymorphism and fracture risk. No significant difference in genotype distribution was found when stratifying by race. When stratifying by fracture type, it was found that vertebral fracture cases had a significantly higher frequency of the PvuII pp genotype than controls in five studies (552 cases and 2350 controls). This meta-analysis suggests a modest but statistically significant association between the ESR1 PvuII pp genotype and vertebral fracture.     

ERα基因PvuⅡ、XbaⅠ和ERβ基因RsaⅠ、AluⅠ酶切多态性与冠心病的相关性研究

目的研究盐城地区雌激素受体α(ERα)基因PvuⅡ、XbaⅠ和雌激素受体β(ERβ)RsaⅠ、AluⅠ酶切多态性与冠状动脉粥样硬化性心脏病(CHD)危险因素的相关性。方法选取124例CHD患者和163例健康体检者分别作为CHD组和对照组,采用酶法测定三酰甘油(TG)和总胆固醇(TC),采用直接法测定高密度脂蛋白(HDL)和低密度脂蛋白(LDL),采用聚合酶链反应-限制片断长度多态性方法分析两组ERα基因PvuⅡ、XbaⅠ和ERβ基因RsaⅠ、AluⅠ酶切多态性。结果 CHD组有吸烟史、有家族史、合并高血压及糖尿病的比率,以及体质量指数、TC、TG、LDL-C水平均明显高于对照组,差异有统计学意义(P0.05);男女之间各指标差异无统计学意义(P0.05)。两组ERα基因PvuⅡ、XbaⅠ和ERβ基因RsaⅠ、AluⅠ基因频率分布与理论分布无差别,均符合Hardy-Weinberg遗传平衡,具有群体代表性。CHD组中,pp、xx、RR、AA基因型最多,PP、XX、rr、aa基因型最少;对照组中,Pp、xx、RR、AA基因型最多,PP、XX、rr、aa基因型最少。CHD组p、x基因分布频率明显高于对照组,差异有统计学意义(P0.05)。结论雌激素基因多态性可能是一个有效的治疗CHD的靶点,p、x基因分布频率可能与CHD危险因素相关。     

Lipid, haemostatic and inflammatory variables in relation to the estrogen receptor alpha (ESR1) PvuII and XbaI gene polymorphisms

BACKGROUND: Estrogen is known to affect lipoprotein metabolism, the haemostatic system and inflammatory markers. Our aim was to determine whether estrogen receptor alpha (ESR1) PvuII and XbaI gene polymorphisms can influence lipid, haemostatic and inflammatory variables in healthy Caucasian women and men of reproductive age. METHODS: 58 healthy women (aged between 18 and 45 years) and 55 healthy men (aged between 21 and 45 years) of reproductive age were enrolled in our study. FSH levels, lipid (total cholesterol, triglyceride, HDL cholesterol, lipoprotein(a), apo A-I, apo B), haemostatic (prothrombin time, activated partial thromboplastin time (aPTT), thrombin time, fibrinogen, factor V, VII, VIII, protein C, protein S, antithrombin III) and inflammatory (CRP) variables were measured on autoanalyzers using commercially available kits. Serum VLDL and LDL cholesterol concentrations were calculated with the equation of Friedewald. The ESR1 PvuII and XbaI genotypes were determined with PCR-RFLP method. RESULTS: In the total group, the ESR1 XbaI GG genotype carriers had significantly higher serum lipoprotein(a) concentrations than the AA or AG genotype carriers. Serum total cholesterol concentrations were significantly higher in healthy women with the PvuII CC genotype than in those with the TT or TC genotypes, whereas healthy women with the GG genotype of the ESR1 XbaI polymorphism had significantly higher serum total cholesterol and LDL cholesterol levels compared to those with the AA or AG genotypes. No other effects of the ESR1 PvuII and XbaI polymorphisms were found on the investigated lipid, haemostatic and inflammatory variables either in the total group or in women and men separately. CONCLUSIONS: The ESR1 PvuII and XbaI gene polymorphisms seem to affect lipoprotein metabolism in healthy subjects of peak reproductive age. However, further studies are needed to determine the molecular mechanisms by which the two polymorphisms could influence serum lipid levels.     

Gender-specific effect of estrogen receptor-1 gene polymorphisms in coronary artery disease and its angiographic severity in Chinese population

BACKGROUND: The role of common polymorphisms of the estrogen receptor-1 in coronary artery disease (CAD) and it association with angiographic severity reminds conflicting in sexes and different races. METHODS: Two-hundred ten angiographically defined Chinese CAD patients and 174 control subjects were enrolled. DNA was obtained and the polymorphisms were analyzed by the polymerase chain reaction. The region containing the PvuII T/C and the XbaI A/G sites was amplified. PCR product was cleaved with the restriction endonucleases. RESULTS: No significant differences in PvuII and XbaI genotype and allele frequencies were noted between the CAD and controls.However, when stratified by gender, we noticed the PvuII genotype and allele frequencies were significantly different between CAD and controls, but in male group only, not in female group. Diabetes, hypertension, high LDL levels and the PvuII CC genotype were independent risk factors for CAD. PvuII CC was associated with the angiographic severity of CAD measuring by the number of diseased vessels as well. For XbaI, no association was found with the CAD susceptibility before and after gender stratification. CONCLUSION: This study revealed a gender-specific effect of PvuII polymorphism in Chinese CAD subjects. PvuII gene polymorphisms affect CAD susceptibility in man only. The PvuII CC is a risk factor for CAD and it is associated with angiographic CAD severity.     

白细胞介素6基因启动子区两个突变位点多态性检测方法的建立

目的建立一种快速检测IL-6基因启动子区-597G／A、-572G／C多态性的双重实时荧光PCR方法。方法采用1对引物，2对荧光标记探针，结合荧光共振能量转移原理和熔点曲线分析技术，检测IL-6基因启动子区-572G／C，-597G／A2个位点多态性。结果用建立的双重实时荧光PCR方法对123名健康查体者进行检测，发现中国汉族人IL-6基因启动子区-572位点有3种基因型，分别为GG，GC，CC型；-597位点仅发现4名为GA型，其余均为GG型，尚未发现从型。结论双重实时荧光PCR法简便快速，与其他方法比较具有准确、经济的特点，适合临床基因快速分型。     

雌激素受体基因多态性与绝经后女性代谢综合征的相关性研究

目的：观察雌激素受体（ER）α和β基因多态与绝经后女性代谢综合征（MS）的关系。方法311例无亲缘关系的绝经后MS患者和231例年龄相匹配的健康对照人群,采用聚合酶链反应-限制性片段长度多态性（PCR-RFLP）技术,检测ERα（XbaⅠ、PvuⅡ）和β基因（RsaⅠ、AluⅠ）多态性,并采用SHEsis在线计算平台进行统计学分析。结果与对照组相比,MS患者体重、BMI、SBP、DBP以及血清FBS、GSP、TG水平显著高于对照组,而HDLC水平显著低于对照组（P＜0．05）;对照组和MS组PP、Pp、pp基因型和P、p等位基因分布频率分别为13．0％、52．8％、34．2％、39．4％、60．6％和9．3％、51．1％、39．5％、34．9％、65．1％,XX、Xx、xx基因型和X、x等位基因分布频率分别为4．3％、48．1％、47．6％、28．4％、71．6％和3．9％、39．2％、56．9％、23．5％、76．5％,AA、Aa、aa基因型和A、a等位基因分布频率分别为73．2％、24．7％、2．2％、85．5％、14．5％和74．0％、24．1％、1．9％、86．0％、14．0％,两组间上述三个酶切位点的基因型及等位基因分布频率均无显著差异（P＞0．05）;对照组和MS组RR、Rr、rr基因型和R、r等位基因分布频率分别为45．0％、44．6％、10．4％、67．3％、32．7％和54．7％、38．9％、6．4％、74．1％、25．9％,MS组RR基因型和R等位基因分布频率显著高于对照组,Rr、rr基因型和r等位基因分布频率显著低于对照组（P＜0．05）;单体型分析显示,R-A单体型在MS组所占的频率显著高于对照组（OR：1．405;95％CI：1．100-1．769;P＜0．05）,而r-A单体型所占频率显著降低（OR：0．700,95％CI：0．536-0．915;P＜0．05）。结论ERα基因多态性与绝经后女性MS无相关性,而ERβRsaⅠ酶切位点基因多态性与绝经后女性MS患者的遗传易感性有关,R等位基因可能是其易感等位基因,而r?     

双重实时荧光PCR与DNA测序分析及PCR—RFLP方法检测白细胞介素-6基因启动子区多态性方法的比较研究

目的比较双重实时荧光PCR与DNA测序分析以及PCR-RFLP三种检测方法对同一样本检测结果的一致性。方法分别采用三种方法对77例样本IL-6基因启动子区-572G／C，-597G／A2个位点多态性进行检测。结果在77例样本中，采用双重实时荧光PCR方法与DNA测序分析相比仅有3例结果不一致。用限制性内切酶BSrBI酶切，有20例与实时荧光PCR方法结果不一致。当PCR产物用限制性内切酶FokI酶切时，仅有5例被发现是GA型，另外两例未被检测出。结论双重实时荧光PCR方法与其它方法比较具有简单、准确、快速的特点，便于临床实验室大规模样本筛壹。