【作者】 王保军；

【导师】 张旭；

【作者基本信息】 华中科技大学 ， 外科学， 2009， 博士

【摘要】 背景原发性醛固酮增多症（简称原醛，Primarv Aldosteronism，PA）是最常见的内分泌高血压之一，两种常见临床亚型为醛固酮瘤（Aldosterone ProducingAdenoma，APA）和特发性醛固酮增多症（Idiopathic Hyperaldosteronism，IHA）。原醛主要的病理生理特点是过多醛固酮的自主分泌。APA多采用手术治疗；尽管大多数原醛患者术后血压恢复正常，但仍有33～44％术后持续性高血压。然而，目前过多醛固酮分泌的机制和术后血压恢复不均一的机制仍不清楚。影响高血压醛固酮代谢和血压调控的遗传因素是否与APA过多醛固酮分泌和术后持续高血压相关还仍不清楚。CYP11B2基因编码合成醛固酮的醛固酮合酶；CYP11B1基因则编码合成皮质醇的11-β羟化酶，两基因位置相近，均位于染色体8q24。研究表明两基因座位上的许多DNA多态性位点处于完全连锁不平衡状态（Linkage Disequilibrium，LD），且CYP11B2基因多态性位点（如rs1799998，intron2野生型／转位型和rs4539）与原发性高血压、11-β羟化酶活性降低、醛固酮分泌与代谢失调，心血管风险密切相关。上述研究提示CYP11B2／B1多态性位点可能与原醛临床表型及术后血压相关。在此假设基础上，本研究对6个DNA多态性位点，包括CYP11B2基因的rs1799998（C-344T）、intron2野生型／转位型、rs4539（A2718G）、rs6414和CYP11B1基因的rs6387（G225A）、rs6410（A2803G）进行基因分型和单体型分析，以判断它们对原醛中CYP11B2基因表达、临床表型的影响，并探讨它们在术后持续高血压中的作用。方法1．采用DNeasy Blood&Tissue试剂盒（Qiagen公司）提取93例随访到的APA患者的组织DNA；在公共SNP数据库（http：／／ncbi．nlm．nih．gov／SNP／和http：／／www．hapmap．org/）中选择CYP11B2和CYP11B1基因中频数≥0．05，且文献报道与醛固酮代谢和高血压相关的位点，包括CYP11B2基因的4个多态性位点：rs1799998、intron2野生型／转位型、rs4539和rs6416及CYP11B1基因的2个多态性位点：rs6410和rs6387。采用一对独立的PCR扩增intron2野生型／转位型位点，其余5个位点采用MGB-Taqman探针法进行基因分型。采用Haploview 4．0分析LD和Hardy-Weinberg平衡。2．采用SYBR Green荧光定量RT-PCR法检测69例APA组织和20例正常肾上腺组织（取白手术的肾癌患者）中CYP11B2和CYP11B1基因的表达，2^-△△CT法计算APA组织中基因表达量相对正常肾上腺组织的变化。放射性免疫法检测血浆醛固酮的浓度（PAC）和血浆肾素活性（PRA），并用Pearson相关分析它们与基因表达的关系。3．在R statistics program 2．7．0程序包中使用SNPassoc 1．5-3和Haplo．stats 13．8分析CYP11B2和CYP11B1基因型及单体型与CYP11B2基因表达量和血浆醛固酮浓度、收缩压、舒张压等临床表型的关系。4．在R statistics program 2．7．0程序包中使用SNPassoc 1．5-3和Haplo．stats 1．3．8分析CYP11B2和CYP11B1基因型及单体型与原醛术后血压恢复的相关性。结果1．检测了6个基因多态性位点，其中5个位点（包括CYP11B2基因的rs1799998、intron2野生型／转位型、rs4539和CYP11B1基因的rs6387、rs6410）在所有随访到的的APA患者中均获得成功检测，等位基因频数均＞0．2，基因型频数均＞0．01。所有位点基因型分布均符合Hardy-Weinberg平衡（对rs6387，rs6410，rs4539，intron2野生型／转位型，rs1799998：P分别为0．06，0．71，1．0，0．12，0．11）。配对的LD分析表明rs6387与rs6410和rs1799998存在着较强的LD（D′0．95，0．73），rs4593和rs1799998及rs6410和intron 2野生型／转位型之间也有稍强的LD（D′0．52）。2．在69例APA和20例正常肾上腺组织中均检测到CYP11B2 mRNA和CYP11B1mRNA的表达。相对正常肾上腺组织，APA组织中CYP11B2 mRNA表达明显上调，为正常肾上腺组织的3．58倍（p＜0．01）。且APA组织中CYP11B2 mRNA表达与血浆醛固酮水平有明显相关性（r=0．649，p＜0．01），但与血浆肾素活性无关（r=-0．133，p=0．277）。APA组织CYP11B1 mRNA表达降低，是正常肾上腺组织的0．54倍，但两者差异无统计学意义（p=0．089）。APA组织中CYP11B1 mRNA表达与血浆肾素活性无相关性（r=-0．165，p=0．185），与血浆醛固酮水平也无相关性（r=-0．08，p=0．946）。3．单个位点分析中，未发现CYP11B2和CYP11B1基因的多态性位点包括rs1799998、intron2野生型／转位型、rs4539、rs410和rs6387与CYP11B2基因mRNA表达，血浆醛固酮浓度，术前收缩压、舒张压相关（p均＞0．05）。单体型分析中，GlobalScore统计显示：CYP11B2-CYP11B1单体型与CYP11B2mRNA表达量增加相关（global-stat=16．18，df=8，p=0．04），与血浆醛固酮水平也相关（global-stat=20．41，df=8，P=0．009）；但与术前收缩压和舒张压不相关（分别P=0．34，P=0．54）。多元回归分析发现单体型H1和H3与CYP11B2基因mRNA的表达上调相关（经过Bonferroni校正后分别p=0．02；0．03），H6、H10和H16与过多的醛固酮分泌相关（经过Bonferroni校正后分别p=0．015；0．003；p=0．049）；但未见有单体型与术前收缩压和舒张压相关（p均＞0．05）。4．单个位点分析：上述五个位点包括rs1799998、intron2野生型／转位型、rs4539、rs410和rs6387中只有rs4539（AA）与术后持续性高血压相关（经过Bonferroni校正后p=0．01）。单体型分析中：Global Score统计显示CYP11B2-CYP11B1单体型与原醛术后血压恢复相关（Max-Stat sim．p=0．006）；多元回归分析中发现单体型H1和H3是预测术后持续性高血压的遗传性危险因子（经过Bonferroni校正后，分别p=0．02；0．03）。另外，传统因素包括高血压持续时间长，有高血压家族史和术前较高的收缩压是术后恢复不好的传统风险因素（经过Bonferroni校正后，分别p=0．002，=0．0015，＜0．00005）。结论1．对随访到的93位APA患者获得了较为完善的CYP11B2和CYP11B1基因多态性的遗传学资料，为后续证实CYP11B2／B1基因多态性和临床表型关系的研究提供了基础。2．APA患者血浆醛固酮的过多分泌可能与CYP11B2基因的表达上调有关，而后者可能与APA组织中出现CYP11B1基因的表达相关。3．尽管作用较弱，本研究显示APA患者CYP11B2和CYP11B1基因多态性可能通过上调CYP11B2的表达，导致血浆醛固酮水平的升高。4．尽管作用较弱，本研究显示APA患者术后血压可能受到CYP11B2／CYP11B1基因多态性的影响。提示遗传因素在APA患者术后血压恢复不均一的机制中可能起一定的作用，单体型H1和H3可能成为预测术后持续性高血压的遗传指标。更多还原

【Abstract】 Primary aldosteronism （PA） is the most common cause of endocrine hypertension.Two common subtypes of PA are idiopathic hyperaldosteronism （IHA） and aldosteroneproducing adenoma （APA）.The main pathophysiological feature of PA is auto-secretion ofexcessive aldosterone.APA are always given surgically correction.Though most patientshad resolution of hypertension after adrenalectomy for PA,postoperative hypertensionoften persisted in a part of patients （account for 33～44%）.However,the mechanisms ofauto-secretion of excessive aldosterone and heterogeneity of BP response to adrenalectomywere not clear nowadays.And,it was not clear whether the genetic variants that influencesteroid synthesis were also associated with auto-secretion of excessive aldosterone andpostoperative hypertension of patients with APA.The terminal stages in the synthesis of steroid are catalyzed by the enzymesaldosterone synthase and 11β-hydroxylase respectively.CYP11B2 gene encodedaldosterone synthase and CYP11B1 gene encodes 11β-hydroxylase.The two genes areclosely adjacent on chromosome 8,band 8q24.The findings of studies demonstrate thatgenotypes at the CYP11B2-CYP11B1 locus are in strong linkage disequilibrium （LD）.Thepolymorphisms of CYP11B2 gene such as rs1799998,intron2 wild/conversion and rs4539were suggested to associate with hypertension and cardiovascular disease risk.Thesesuggestion cued these genetic variations within CYP11B2/B1 might exert deleterious effect on clinical phenotype and postoperative resolution of hypertension for PA.On the basis ofthe above hypothesis,six DNA Polymorphisms at CYP11B2/CYP11B1 locus,includingrs1799998 （C-344T）,intron2 wild/conversion （w/c）,rs4539 （A2718G） and rs6414 ofCYP11B2 as well as rs6410 （G225A） and rs6387 （A2803G） of CYP11B1 were genotyedand analysed in the 93 follow-up patients with PA.The aim of this stduy was to investigatethe role of these polymorphisms/haplotype in CYP11B2 gene mRNA,clinical phenotype,and postoperative resolution of hypertension.METHODS1.DNA was extracted from tumorous tissue samples of 93 follow-up patients withAPA according to the manufacturer instructions with DNeasy Blood & Tissue Kit （Qiagen,Cat.No.69504,Germany）.DNA polymorphisms at CYP11B2/CYP11B1 locus weresearched in public databases （http://ncbi.nlm.nih.gov/SNP/ and http://www.hapmap.org/）.Selected potential loci had a minor allele frequency =0.05 based on data of Japanese andChinese population.In addition,previous studies produced the evidence of associationsbetween these variations and blood pressure regulation.Thus,above-mentioned six DNApolymorphisms including five single-nucleotide polymorphisms （SNPs） and intron2 w/cpolymorphism of CYP11B2 were included in the study.CYP11B2 intron 2 w/c wasgenotyped by two separate PCRs as described previously.And five SNPs were detected byTaqman SNP genotyping assays.Pairwise LD and Hardy-Weinberg equilibrium wasdetermined with Haploview 4.0.LD was estimated by D’.2.The expressions of CYP11B1 and CYP11B2 gene were examined by SYBR Greenreal-time RT-PCR assay with 2^-△△C_T method in a series of adrenal tissues,including 69tumorous tissues of patients with APA and 20 normal adrenals from patientsadrenalectomized for renal cancer.PAC and PRA were measured by radioimmunoassaytechnique,and their associations with two above genes were analysed by Pearsoncorrelation.3.The analysis of associations between polymorphisms at CYP11B1/CYP11B2 geneand clinical phenotypes or mRNA expression of CYP11B2 gene was performed withSNPassoc 1.5-3 for R statistics program 2.7.0.And the analysis of association betweenhaplotype and clinical phenotypes or mRNA expression was performed with Haplo.stats 1.3.8 for R statistics program 2.7.0.4.The analysis of association between polymorphisms and postoperativehypertension was performed with SNPassoc 1.5-3 for R statistics program 2.7.0.And theanalysis of association between haplotype and postoperative hypertension was performedwith Haplo.stats 1.3.8 for R statistics program 2.7.0.RESULTS1.A total of 5 potentially genetic markers,including rs1799998,intron 2 w/c,andrs4539 within CYP11B2 and,rs6410 and rs6387 within CYP11B1 gene,were detected in93 follow-up patients with APA.It obtained full genotype data from 93 patients.The allelefrequencies （＞0.2） and Genotype frequencies （＞0.01） of the 5 genetic markers were seenhere.No marker exhibited a significant difference between Hardy-Weinberg equilibrium（for rs6387,rs6410,rs4539,intron2 w/c,rs1799998;P=0.06,0.71,1.0,0.12,0.11respectively）.Pairwise LD was shown that rs6387 was in strong LD with rs6410 andrs1799998 （D’ 0.95,0.73）.Mild LD was also seen between rs4593 and rs1799998 （D’ 0.50）as well as between rs6410 and intron 2 w/c （D’ 0.52）.2.The expressions of CYP11B1 and CYP11B2 gene were detected in all the tissues.Compared with that in normal adrenal tissue,the mRNA expression of CYP11B2 gene inAPA tissue up-regulated significantly （as 3.58 times as that in normal adrenal tissue,p＜0.01）.And this increase of mRNA expression in APA was correlated with PAC （r=0.649,p＜0.01）,but not with PRA （r=-0.133,p-0.277）.The expression of CYP11B1 in APA tissuedown-regulated in APA （as 0.54 times as that in normal adrenal tissue）,but it had nosignificence in statistics （p=0.089）.The decreased expression of CYP11B1 has nevercorrelation with PRA （r=-0.165,p= 0.185） or PAC （r=-0.08,p= 0.946）.3.In the analysis of single locus,none of the five polymorphisms includingrs1799998,intron2w/c,rs4539,rs6410 and rs6387 were correlated with mRNA expressionsof CYP11B2,clinical phenotype including PAC,SBP and DBP （allp＞0.05）.In the analysisof haplotype,global score statistics indicated CYP11B2-CYP11B1 haplotype wasassociated with mRNA expression of CYP11B2 （global-stat=16.18,df=8,p=0.04） and PAC（global-stat = 20.41,df = 8,p= 0.009）,but not with SBP and DBP（p=0.34,p=0.54）.Inregression models of haplo.glm,it was found that haplotype H1 and H3 were associated with upregulated mRNA expression of CYP11B2 （p=0.02;0.03 respectively afterBonferroni correction）,while H6,H10 and H16 associated with PAC （p=0.015;0.003,0.049 respectively after Bonferroni correction）.However none haplotype was foundassociation with SBP or DBP （allp＞0.05）.4.In the analysis of single locus,we found only rs4539AA of the five polymorphismsincluding rs1799998,intron2 wild/conversion,rs4539,rs6410and rs6387 were correlatedwith postoperative hypertension of patients with APA （p=0.01 after Bonferroni correction）.In the analysis of haplotype,global score statistics indicated CYP11B2-CYP11B1haplotype was associated with postoperative hypertension （Max-Stat sim.p=0.006）.Inregression models of haplo.glm,it was found that haplotype H1 and H3 were geneticpredictors for postoperative persisting hypertension of APA （p=0.02,p=0.03 respectivelyafter Bonferroni correction）.In addition,the traditional risk factors such as duration ofpreoperative hypertension,family history of hypertension and preoperative higher level ofSBP were also identified for postoperative persisting hypertension （p=0.002,0.0015,＜0.00005 respectively after Bonferroni correction）.CONCLUSIONS1.The full genotype data from 93 patients with APA provided help and basis for thesubsequent association study between genetic variation and clinical phenotype.2.Excessive secretion of PAC in APA patients may correlated with up-regulatedmRNA expression of CYP11B2,whose behavior was related with abnormal mRNAexpression of CYP11B1 in APA tissue.3.DNA polymorphisms at CYP11B1/CYP11B2 gene might impact PAC throughupregulation of mRNA expression of CYP11B2 gene in APA.4.Postoperative persisting hypertension of patients with APA might be influenced byDNA Polymorphisms at CYP11B2/CYP11B1 locus.Genetic effect might play a role inheterogeneity of BP response to adrenalectomy.And H1 and H3 might be genetic predictorsfor postoperative persisting hypertension of APA.更多还原