醛固酮合酶和11-β羟化酶基因多态性与原发性醛固酮增多症发病风险的相关性研究
摘要
背景
原发性醛固酮增多症(Primary aldosteronism，PA)是最常见的继发性高血压(Secondary Hypertension，SH)病因之一，以醛固酮的非抑制性自主高分泌紊乱为特征，可以引起低肾素、低血钾、钠水潴留和高血压等症候群。醛固酮瘤(Aldosterone-Producing Adenoma，APA)是PA最常见的亚型，约占PA 60％，其次是特发性醛固酮增多症(Idiopathic Hyperaldosteronism，IHA)，约占PA 30％。醛固酮(Aldodosterone，ALD)在PA的发生、发展过程中扮演着非常重要的角色。ALD合成的最后关键步骤包括：脱氧皮质酮向皮质酮转化的11-β羟化反应、皮质酮转化为18-羟皮质酮的18-羟化反应和18-羟皮质酮转化为ALD的18-氧化反应。上述三个步骤均由醛固酮合酶(Aldosterone synthase)催化。编码该酶的基因是CYP11B2。在肾上腺皮质的束状带，11-脱氧皮质醇在11-β羟化酶的作用下产生皮质醇，编码11-β羟化酶的基因是CYP11B1。研究发现，CYP11B2和CYP11B1的几个常见的多态性位点(如：-344T／C、intron 2W／C、K173R、2803A／G等)与原发性高血压(Essential Hypertension，EH)相关，并影响ALD的合成。近年来，有关CYP11B2基因多态性与PA相关性研究的报道较少，且多为小样本研究。另外，尚未见到CYP11B1基因多态性与PA相关性研究的报道。
方法
1．收集118例正常人血液标本，134例APA患者肾上腺皮质腺瘤标本(88个冰冻标本和46个石蜡标本)和45例IHA患者肾上腺标本(31个冰冻标本和14个石蜡标本)。采用DNeasy Blood & Tissue试剂盒(Qiagen公司)提取标本DNA，-20℃保存。
2．根据相关文献和公共SNP数据库(http：／／ncbi．nlm．nih．gov／SNP／和http：／／www．hapmap．org／)选择CYP11B2和CYP11B1基因中频数≥0．05、且文献报道与高血压病相关的6个位点(rs1799998、rs4539、rs6414、intron 2W／C、rs6410和rs6387)。
3．对CYP11B2和CYP11B1基因的6个多态性位点进行检测。其中，intron 2W／C采用2个独立的PCR反应，其余位点均先后采用：聚合酶链式反应-限制性片段长度多态性(Polymerase Chain Reaction-Restriction Fragment Length PolymorphismPCR-RFLP)、MGB-Taqman探针法和测序法。并采用Haploview 4．0分析连锁不平衡(Linkage Disequilibrium，LD)，在R statistics program 2．7．0程序包中使用SNPassoc1．5-3分析Hardy-Weinberg平衡。
4．在R statistics program 2．7．0程序包中使用SNPassoc 1．5-3和Haplo．stats 1．3．8软件分析CYP11B2和CYP11B1基因多态性位点与PA的关系。
结果
1．基因型鉴定：本研究所选择的6个双等位基因多态性位点除rs6414外，均获得成功检测。各基因型分布均符合Hardy-Weinberg平衡(P＞0．05)。rs6410和rs6387位点之间的LD最为强烈(D’0．66)，LD最弱的是rs4539和intron 2 W／C位点(D’0．06)。
2．比较PCR-RFLP法和MGB-Taqman探针法的优缺点：检测rs1799998和rs4539多态性位点时，MGB-Taqman探针法的误差率明显低于PCR-RFLP法(x~2=7．39，P=0．007；x~2=6．34，P=0．012)。采用MGB-Taqman探针法检测rs1799998和rs4539位点平均所用时间(1h左右)较PCR-RFLP法(6h左右)短，但其费用(15．00元人民币左右)较PCR-RFLP法(rs1799998位点为6．00，rs4539位点为12．00元人民币左右)高。
3．CYP11B2和CYP11B1基因型和PA的关系：APA和IHA组中rs6410位点的A等位基因频数显著高于对照组(P=1．09×10~(-5)，P=0．015)。与对照组相比，APA组中rs6410位点纯合子AA基因型和杂合子AG基因型比例较高(P=2．19×10~(-4))。与对照组相比，IHA组中纯合子AA基因型和杂合子AG基因型频数仅在年龄、性别和BMI校正后具有统计学意义(OR=4．06 95％CI 1．31-12．66；OR=2．41，95％CI 1．02-5．72)。本研究中，未发现rs4539、intron 2 W／C和rs1799998单个多态性位点与APA和IHA相关。
4．CYP11B2和CYP11B1单体型和APA的关系：与对照组相比，AAAWT单体型频数在APA中显著增高(Empirical P=5．0×10~(-5))。相反，GGAWT单体型频数在APA中显著降低(Empirical P=1．0×10~(-4))。以年龄、性别和BMI进行校正后，发现AAAWT单体型与APA的发病风险增高相关(OR=1．44，95％CI 1．19-1．76)，而GGAWT单体型则与APA的发病危险性降低相关(OR=0．73，95％CI 0．55-0．97)。
5．CYP11B2-CYP11B1单体型与IHA的关系：与对照组相比，IHA组单体型AAAWT频数增高(P=0．002)。以年龄、性别和BMI进行校正后，发现AAAWT，AGGWT和AGAWC单体型与IHA的发病危险性增高相关(OR=1．55，95％CI 1．23-1．96；OR=1．49，95％CI 1．17-1．89；OR=1．40，95％CI 1．04-1．88)。而且，AAAWT在IHA中增高的幅度大于在APA中增高的幅度(OR=1．55比OR=1．44)。
结论
1．在选择基因多态性检测方法时，应该根据所选位点、样本量大小和实验室经费等情况综合考虑，选择最为合适的检测方法。MGB-Taqman探针法耗时少，效率高，准确性强。适用于大批量基因多态性检测。PCR-RFLP法适应于样本量小，内切酶价格低的多态性位点检测。
2．CYP11B2和CYP11B1基因多态性与原发性醛固酮增多症的发病显著相关。通过对二者多态性位点的检测，有可能为预测原发性醛固酮增多症的发病提供一定的参考价值。
3．本研究为进一步研究原发性醛固酮增多症的发病机制以及对其开展基因治疗提供了新的线索和思路。
Introduction
Primary aldosteronism (PA) is now recognized as the most common form ofsecondary hypertension.Cross-sectional and prospective studies reported PA in more than10% of hypertensive patients,both in general and in specialty settings.The most commonclinical subtypes of primary aldosteronism are aldosterone-producing adenoma (APA),occurring in about 60% of cases,and idiopathic hyperaldosteronism (IHA) accounting forabout 30% of cases.Aldosterone plays a key role in APA,and the terminal stages in itssynthesis,the synthesis of aldosterone from l l-deoxycorticosterone in the zoneglomerulosa,arecatalyzed by aldosterone synthase,which is encoded by the CYP11B2gene.Parallel 11β-hydroxylation of the 17-hydroxysteroid,by the enzyme11β-hydroxylase encoded by the gene CYP11B1,produces cortisol in the zona fasciculata.CYP11B2 and CYPllB1 are situated approximately 40 kilobases apart,on chromosome 8,band 8q24 in man.Several frequent polymorphisms (E.g.-344T/C,intron 2 W/C andK173R) in CYP11B2 are suggested to have associations with essential hypertension andmay influence aldosterone secretion.However,there was few small sample of investigationin the genetic association between the CYP11B2 and CYP11B1 polymorphisms and thedevelopment of PA.No investigation in the association between the CYP11B1polymorphisms and the development of PA.In this study,we aimed to identifypolymorphisms in CYP11B2 and CYP11B1 genes that are associated with PA,with thehypothesis that genetic variants in them may contribute to PA.
Methods
1.Among the participants,119 cases (88 with APA and 31 with IHA) provided frozentissues,60 cases (46 with APA and 14 with IHA) provided paraffin-imbedded tissues and118 controls provided peripheral blood samples for genotyping.DNA was extracted fromthe tissues and peripheral blood using DNeasy Blood & Tissue Kit (Qiagen,Cat.No.69504,Germany) as instructed,and maintained at -20℃.
2.For the CYP11B2 and CYP11B1 genes,6 polymorphisms of minor allele frequency≥0.05 were selected in public databases (http://ncbi.nlm.nih.gov/SNP/ andhttp://www.hapmap.org/) for study on the basis of evidence suggesting functional relevanceor reports of association with hypertension.Three SNPs span the CYP11B2 gene:rs1799998 in the promoter region (also known in previous publications as C-344T or SF-1),rs4539 in the third exon (also known as A2718G or K173R) and rs6414 in the third intron.Two SNPs span the CYP11B1 gene:rs 6410 (G225A) in the first exon and rs6387(A2803G) in the third intron.We also typed the biallelic intron 2W/C conversionpolymorphism in the CYP11B2 gene.
3.Analysis of the intron 2W/C conversion polymorphism was performed by use of 2separate PCRs.The other polymorphisms of CYP11B2 and CYP11B1 genes were detectedusing the Polymerase Chain Reaction-Restriction Fragment Length Polymorphism(PCR-RFLP,MGB-Taqman probs and DNA sequencing methods in turn.With the help ofHaploview 4.0,pairwise LD was estimated by D',and difference of allelic frequenciesbetween cases and controls of each polymorphism and allelic permutation test with 10,000times were performed.Hardy-Weinberg equilibrium were determined by SNPassoc 1.5-3 inR statistics program 2.7.0.
4.The genotype frequencies between cases and controls of each polymorphism weredetermined by SNPassoc 1.5-3.Haplotype frequencies for various polymorphismcombinations were estimated by Haplo.stats 1.3.8.The differences in haplotype frequencyprofiles between the case and control groups,and haplotype-based hypothesis tests ofgeneralized linear models were conducted using the software Haplo.stats 1.3.8.
Results
1.Genotyping:All the selected diallelic polymorphisms except rs6414 in thecase-control populations were successfully genetyped.The distribution of genotypes was in accordance with Hardy-Weinberg Equilibrium in each group (P＞0.05).The degrees ofpairwise LD among all selected variants were estimated by the standardized disequilibriumcoefficient D'.It was shown that these 5 variants are in different strength of LD,with thers6410 and rs6387 polymorphism in the tightest LD (D' 0.66),and rs4539 and intron 2 W/Chaving the weakest LD (D' 0.06) in control subjects.
2.Comparison of PCR-RFLP and MGB-Taqman probe:The error rate ofMGB-Taqman probe was significantly lower than that of PCR-RFLP for detectingrs1799998 and rs4539 (χ~2=7.39,P=0.007;χ~2=6.34,P=0.012;respectively).The averagetime for detecting rs1799998 and rs4539 by PCR-RFLP was about 6h,and byMGB-Taqman probe was 1 h.The average costs for detecting rs1799998 and rs4539 byPCR-RFLP were 6.00 and 12.00 yuan,respectively,and by MGB-Taqman probe were15.00 yuan.
3.CYP11B2 - CYP11B1 genotypes in relation to PA:Univariate allelic frequencyanalyses revealed that only rs6410 was significantly associated with APA and IHA atP=1.09×10~(-5) and P=0.015,respectively.There was a relative excess of AA homozygotesand AG heterozygotes of rs6410 allele in APA group compared with control group (P=2.19×10~(-4)).Multivariate unconditional logistic regression analyses demonstrated that thissignificant association between rs6410 and APA was consistent even when adjusted for age,gender,and body mass index (BMI) (P＜0.05).There were significantly different genotypesAA and AG of rs6410 allele between patients with IHA and controls only after adjusted forage,gender,and BMI (OR=4.06 95% CI 1.31-12.66;OR=2.41,95% CI 1.02-5.72.Incontrast,there was no significant difference in rs4539,Intron 2W/C and rs1799998 betweencontrol group and APA or IHA group.
4.CYP11B2 -CYP11B1 haplotypes in relation to APA:A highly significant differencewas observed in the haplotype frequencies between the control subjects and the patientswith APA (Global-stat = 31.95,df = 9,P = 0.0002).Individually,the susceptible haplotypeAAAWT was identified to be significantly associated with APA with empirical P values of5.0×10~(-5).In contrast,one protective haplotype GGAWT showed significant differencebetween patients with APA and controls (Empirical P=1.0×10~(-4)).The susceptible haplotypeAAAWT displayed a significantly increased risk for APA (odds ratio 15.89,95% CI2.06-122.89),which was consistent even when adjusted for age,gender and BMI (odds ratio 1.44,95% CI 1.19-1.76).In contrast,the protective haplotype GGAWT exhibitedsignificantly decreased effect on APA (odds ratio 0.08,95% CI 0.01-0.69).In addition,twosusceptible haplotypes GAACT and AGGWT were found with a marginal significance inthe difference between control group and APA group after adjusted for age,gender andBMI (odds ratio 1.28,95% CI 0.97-1.71 and odds ratio 1.23,95% CI 0.99-1.54,respectively).
5.CYP11B2 -CYP11B1 haplotypes in relation to IHA:A significant difference wasfound in the haplotype frequencies between the control subjects and the patients with IHA(Global-stat=19.06,df =9,P = 0.025).Interestingly,the haplotype AAAWT was alsoidentified to be significantly associated with IHA (P=0.002).The subjects carryingsusceptible haplotype AAAWT showed a significant increased risk for IHA (odds ratio4.99,95% CI 1.29-19.35),which was consistent even when adjusted for age,gender andBMI (odds ratio 1.55,95% CI 1.23-1.96).A susceptible haplotype AGGWT was foundwith a marginal significance in the difference between control group and IHA group (oddsratio 2.92,95% CI 0.82-10.45),and had a significantly increased risk after adjusted for age,gender and BMI (odds ratio 1.49,95% CI 1.17-1.89).Another susceptible haplotypeAGAWC was found to have a significantly increased risk only after adjusted for age,gender and BMI (odds ratio 1.40,95% CI 1.04-1.88).
Conclusions
1.The most appropriate method should be selected to detect the DNA polymorphismsaccording to the selected polymorphisms,the number of samples and the cost which youcan provide.MGB-Taqman probe could be used to detect the polymorphisms of largersamples for its less time-consuming,efficiency and accuracy.PCR-RFLP was suitable todetecting the smaller sample and those polymorphisms,the incision enzymes of which wereinexpensive.
2.Our results reveal highly significant association between genetic variations inCYP11B2 and CYP11B1 genes and genetic predisposition to PA.PA may be prognosed bydetecting the polymorphisms of CYP11B2 and CYP11B1 genes.
3.Our study maybe provided a way to investigate the pathogenesy and gene therapy of PAKeywords:primary aldosteronism;CYP11B2;CYP11B1;polymorphism
原发性醛固酮增多症(Primary aldosteronism，PA)是最常见的继发性高血压(Secondary Hypertension，SH)病因之一，以醛固酮的非抑制性自主高分泌紊乱为特征，可以引起低肾素、低血钾、钠水潴留和高血压等症候群。醛固酮瘤(Aldosterone-Producing Adenoma，APA)是PA最常见的亚型，约占PA 60％，其次是特发性醛固酮增多症(Idiopathic Hyperaldosteronism，IHA)，约占PA 30％。醛固酮(Aldodosterone，ALD)在PA的发生、发展过程中扮演着非常重要的角色。ALD合成的最后关键步骤包括：脱氧皮质酮向皮质酮转化的11-β羟化反应、皮质酮转化为18-羟皮质酮的18-羟化反应和18-羟皮质酮转化为ALD的18-氧化反应。上述三个步骤均由醛固酮合酶(Aldosterone synthase)催化。编码该酶的基因是CYP11B2。在肾上腺皮质的束状带，11-脱氧皮质醇在11-β羟化酶的作用下产生皮质醇，编码11-β羟化酶的基因是CYP11B1。研究发现，CYP11B2和CYP11B1的几个常见的多态性位点(如：-344T／C、intron 2W／C、K173R、2803A／G等)与原发性高血压(Essential Hypertension，EH)相关，并影响ALD的合成。近年来，有关CYP11B2基因多态性与PA相关性研究的报道较少，且多为小样本研究。另外，尚未见到CYP11B1基因多态性与PA相关性研究的报道。
方法
1．收集118例正常人血液标本，134例APA患者肾上腺皮质腺瘤标本(88个冰冻标本和46个石蜡标本)和45例IHA患者肾上腺标本(31个冰冻标本和14个石蜡标本)。采用DNeasy Blood & Tissue试剂盒(Qiagen公司)提取标本DNA，-20℃保存。
2．根据相关文献和公共SNP数据库(http：／／ncbi．nlm．nih．gov／SNP／和http：／／www．hapmap．org／)选择CYP11B2和CYP11B1基因中频数≥0．05、且文献报道与高血压病相关的6个位点(rs1799998、rs4539、rs6414、intron 2W／C、rs6410和rs6387)。
3．对CYP11B2和CYP11B1基因的6个多态性位点进行检测。其中，intron 2W／C采用2个独立的PCR反应，其余位点均先后采用：聚合酶链式反应-限制性片段长度多态性(Polymerase Chain Reaction-Restriction Fragment Length PolymorphismPCR-RFLP)、MGB-Taqman探针法和测序法。并采用Haploview 4．0分析连锁不平衡(Linkage Disequilibrium，LD)，在R statistics program 2．7．0程序包中使用SNPassoc1．5-3分析Hardy-Weinberg平衡。
4．在R statistics program 2．7．0程序包中使用SNPassoc 1．5-3和Haplo．stats 1．3．8软件分析CYP11B2和CYP11B1基因多态性位点与PA的关系。
结果
1．基因型鉴定：本研究所选择的6个双等位基因多态性位点除rs6414外，均获得成功检测。各基因型分布均符合Hardy-Weinberg平衡(P＞0．05)。rs6410和rs6387位点之间的LD最为强烈(D’0．66)，LD最弱的是rs4539和intron 2 W／C位点(D’0．06)。
2．比较PCR-RFLP法和MGB-Taqman探针法的优缺点：检测rs1799998和rs4539多态性位点时，MGB-Taqman探针法的误差率明显低于PCR-RFLP法(x~2=7．39，P=0．007；x~2=6．34，P=0．012)。采用MGB-Taqman探针法检测rs1799998和rs4539位点平均所用时间(1h左右)较PCR-RFLP法(6h左右)短，但其费用(15．00元人民币左右)较PCR-RFLP法(rs1799998位点为6．00，rs4539位点为12．00元人民币左右)高。
3．CYP11B2和CYP11B1基因型和PA的关系：APA和IHA组中rs6410位点的A等位基因频数显著高于对照组(P=1．09×10~(-5)，P=0．015)。与对照组相比，APA组中rs6410位点纯合子AA基因型和杂合子AG基因型比例较高(P=2．19×10~(-4))。与对照组相比，IHA组中纯合子AA基因型和杂合子AG基因型频数仅在年龄、性别和BMI校正后具有统计学意义(OR=4．06 95％CI 1．31-12．66；OR=2．41，95％CI 1．02-5．72)。本研究中，未发现rs4539、intron 2 W／C和rs1799998单个多态性位点与APA和IHA相关。
4．CYP11B2和CYP11B1单体型和APA的关系：与对照组相比，AAAWT单体型频数在APA中显著增高(Empirical P=5．0×10~(-5))。相反，GGAWT单体型频数在APA中显著降低(Empirical P=1．0×10~(-4))。以年龄、性别和BMI进行校正后，发现AAAWT单体型与APA的发病风险增高相关(OR=1．44，95％CI 1．19-1．76)，而GGAWT单体型则与APA的发病危险性降低相关(OR=0．73，95％CI 0．55-0．97)。
5．CYP11B2-CYP11B1单体型与IHA的关系：与对照组相比，IHA组单体型AAAWT频数增高(P=0．002)。以年龄、性别和BMI进行校正后，发现AAAWT，AGGWT和AGAWC单体型与IHA的发病危险性增高相关(OR=1．55，95％CI 1．23-1．96；OR=1．49，95％CI 1．17-1．89；OR=1．40，95％CI 1．04-1．88)。而且，AAAWT在IHA中增高的幅度大于在APA中增高的幅度(OR=1．55比OR=1．44)。
结论
1．在选择基因多态性检测方法时，应该根据所选位点、样本量大小和实验室经费等情况综合考虑，选择最为合适的检测方法。MGB-Taqman探针法耗时少，效率高，准确性强。适用于大批量基因多态性检测。PCR-RFLP法适应于样本量小，内切酶价格低的多态性位点检测。
2．CYP11B2和CYP11B1基因多态性与原发性醛固酮增多症的发病显著相关。通过对二者多态性位点的检测，有可能为预测原发性醛固酮增多症的发病提供一定的参考价值。
3．本研究为进一步研究原发性醛固酮增多症的发病机制以及对其开展基因治疗提供了新的线索和思路。
Introduction
Primary aldosteronism (PA) is now recognized as the most common form ofsecondary hypertension.Cross-sectional and prospective studies reported PA in more than10% of hypertensive patients,both in general and in specialty settings.The most commonclinical subtypes of primary aldosteronism are aldosterone-producing adenoma (APA),occurring in about 60% of cases,and idiopathic hyperaldosteronism (IHA) accounting forabout 30% of cases.Aldosterone plays a key role in APA,and the terminal stages in itssynthesis,the synthesis of aldosterone from l l-deoxycorticosterone in the zoneglomerulosa,arecatalyzed by aldosterone synthase,which is encoded by the CYP11B2gene.Parallel 11β-hydroxylation of the 17-hydroxysteroid,by the enzyme11β-hydroxylase encoded by the gene CYP11B1,produces cortisol in the zona fasciculata.CYP11B2 and CYPllB1 are situated approximately 40 kilobases apart,on chromosome 8,band 8q24 in man.Several frequent polymorphisms (E.g.-344T/C,intron 2 W/C andK173R) in CYP11B2 are suggested to have associations with essential hypertension andmay influence aldosterone secretion.However,there was few small sample of investigationin the genetic association between the CYP11B2 and CYP11B1 polymorphisms and thedevelopment of PA.No investigation in the association between the CYP11B1polymorphisms and the development of PA.In this study,we aimed to identifypolymorphisms in CYP11B2 and CYP11B1 genes that are associated with PA,with thehypothesis that genetic variants in them may contribute to PA.
Methods
1.Among the participants,119 cases (88 with APA and 31 with IHA) provided frozentissues,60 cases (46 with APA and 14 with IHA) provided paraffin-imbedded tissues and118 controls provided peripheral blood samples for genotyping.DNA was extracted fromthe tissues and peripheral blood using DNeasy Blood & Tissue Kit (Qiagen,Cat.No.69504,Germany) as instructed,and maintained at -20℃.
2.For the CYP11B2 and CYP11B1 genes,6 polymorphisms of minor allele frequency≥0.05 were selected in public databases (http://ncbi.nlm.nih.gov/SNP/ andhttp://www.hapmap.org/) for study on the basis of evidence suggesting functional relevanceor reports of association with hypertension.Three SNPs span the CYP11B2 gene:rs1799998 in the promoter region (also known in previous publications as C-344T or SF-1),rs4539 in the third exon (also known as A2718G or K173R) and rs6414 in the third intron.Two SNPs span the CYP11B1 gene:rs 6410 (G225A) in the first exon and rs6387(A2803G) in the third intron.We also typed the biallelic intron 2W/C conversionpolymorphism in the CYP11B2 gene.
3.Analysis of the intron 2W/C conversion polymorphism was performed by use of 2separate PCRs.The other polymorphisms of CYP11B2 and CYP11B1 genes were detectedusing the Polymerase Chain Reaction-Restriction Fragment Length Polymorphism(PCR-RFLP,MGB-Taqman probs and DNA sequencing methods in turn.With the help ofHaploview 4.0,pairwise LD was estimated by D',and difference of allelic frequenciesbetween cases and controls of each polymorphism and allelic permutation test with 10,000times were performed.Hardy-Weinberg equilibrium were determined by SNPassoc 1.5-3 inR statistics program 2.7.0.
4.The genotype frequencies between cases and controls of each polymorphism weredetermined by SNPassoc 1.5-3.Haplotype frequencies for various polymorphismcombinations were estimated by Haplo.stats 1.3.8.The differences in haplotype frequencyprofiles between the case and control groups,and haplotype-based hypothesis tests ofgeneralized linear models were conducted using the software Haplo.stats 1.3.8.
Results
1.Genotyping:All the selected diallelic polymorphisms except rs6414 in thecase-control populations were successfully genetyped.The distribution of genotypes was in accordance with Hardy-Weinberg Equilibrium in each group (P＞0.05).The degrees ofpairwise LD among all selected variants were estimated by the standardized disequilibriumcoefficient D'.It was shown that these 5 variants are in different strength of LD,with thers6410 and rs6387 polymorphism in the tightest LD (D' 0.66),and rs4539 and intron 2 W/Chaving the weakest LD (D' 0.06) in control subjects.
2.Comparison of PCR-RFLP and MGB-Taqman probe:The error rate ofMGB-Taqman probe was significantly lower than that of PCR-RFLP for detectingrs1799998 and rs4539 (χ~2=7.39,P=0.007;χ~2=6.34,P=0.012;respectively).The averagetime for detecting rs1799998 and rs4539 by PCR-RFLP was about 6h,and byMGB-Taqman probe was 1 h.The average costs for detecting rs1799998 and rs4539 byPCR-RFLP were 6.00 and 12.00 yuan,respectively,and by MGB-Taqman probe were15.00 yuan.
3.CYP11B2 - CYP11B1 genotypes in relation to PA:Univariate allelic frequencyanalyses revealed that only rs6410 was significantly associated with APA and IHA atP=1.09×10~(-5) and P=0.015,respectively.There was a relative excess of AA homozygotesand AG heterozygotes of rs6410 allele in APA group compared with control group (P=2.19×10~(-4)).Multivariate unconditional logistic regression analyses demonstrated that thissignificant association between rs6410 and APA was consistent even when adjusted for age,gender,and body mass index (BMI) (P＜0.05).There were significantly different genotypesAA and AG of rs6410 allele between patients with IHA and controls only after adjusted forage,gender,and BMI (OR=4.06 95% CI 1.31-12.66;OR=2.41,95% CI 1.02-5.72.Incontrast,there was no significant difference in rs4539,Intron 2W/C and rs1799998 betweencontrol group and APA or IHA group.
4.CYP11B2 -CYP11B1 haplotypes in relation to APA:A highly significant differencewas observed in the haplotype frequencies between the control subjects and the patientswith APA (Global-stat = 31.95,df = 9,P = 0.0002).Individually,the susceptible haplotypeAAAWT was identified to be significantly associated with APA with empirical P values of5.0×10~(-5).In contrast,one protective haplotype GGAWT showed significant differencebetween patients with APA and controls (Empirical P=1.0×10~(-4)).The susceptible haplotypeAAAWT displayed a significantly increased risk for APA (odds ratio 15.89,95% CI2.06-122.89),which was consistent even when adjusted for age,gender and BMI (odds ratio 1.44,95% CI 1.19-1.76).In contrast,the protective haplotype GGAWT exhibitedsignificantly decreased effect on APA (odds ratio 0.08,95% CI 0.01-0.69).In addition,twosusceptible haplotypes GAACT and AGGWT were found with a marginal significance inthe difference between control group and APA group after adjusted for age,gender andBMI (odds ratio 1.28,95% CI 0.97-1.71 and odds ratio 1.23,95% CI 0.99-1.54,respectively).
5.CYP11B2 -CYP11B1 haplotypes in relation to IHA:A significant difference wasfound in the haplotype frequencies between the control subjects and the patients with IHA(Global-stat=19.06,df =9,P = 0.025).Interestingly,the haplotype AAAWT was alsoidentified to be significantly associated with IHA (P=0.002).The subjects carryingsusceptible haplotype AAAWT showed a significant increased risk for IHA (odds ratio4.99,95% CI 1.29-19.35),which was consistent even when adjusted for age,gender andBMI (odds ratio 1.55,95% CI 1.23-1.96).A susceptible haplotype AGGWT was foundwith a marginal significance in the difference between control group and IHA group (oddsratio 2.92,95% CI 0.82-10.45),and had a significantly increased risk after adjusted for age,gender and BMI (odds ratio 1.49,95% CI 1.17-1.89).Another susceptible haplotypeAGAWC was found to have a significantly increased risk only after adjusted for age,gender and BMI (odds ratio 1.40,95% CI 1.04-1.88).
Conclusions
1.The most appropriate method should be selected to detect the DNA polymorphismsaccording to the selected polymorphisms,the number of samples and the cost which youcan provide.MGB-Taqman probe could be used to detect the polymorphisms of largersamples for its less time-consuming,efficiency and accuracy.PCR-RFLP was suitable todetecting the smaller sample and those polymorphisms,the incision enzymes of which wereinexpensive.
2.Our results reveal highly significant association between genetic variations inCYP11B2 and CYP11B1 genes and genetic predisposition to PA.PA may be prognosed bydetecting the polymorphisms of CYP11B2 and CYP11B1 genes.
3.Our study maybe provided a way to investigate the pathogenesy and gene therapy of PAKeywords:primary aldosteronism;CYP11B2;CYP11B1;polymorphism
引文
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9. Wagner MJ, Ge Y, Siciliano M, Wells DE. A hybrid cell mapping panel for regional localization of probes to human chromosome 8. Genomics, 1991,10:114-25.
10. Hautanena A, Lankinen L, Kupari M et al. Associations between aldosterone synthase gene polymorphism and the adrenocortical function in males. J Intern Med, 1998,244:11-8.
11. Barbato A, Russo P, Siani A et al. Aldosterone synthase gene (CYP11B2) C-344T polymorphism, plasma aldosterone, renin activity and blood pressure in a multi-ethnic population. J Hypertens, 2004, 22:1895-901.
12. Mayosi BM, Keavney B, Watkins H, Farrall M. Measured haplotype analysis of the aldosterone synthase gene and heart size. Eur J Hum Genet, 2003,11:395-401.
13. Clyne CD, Zhang Y, Slutsker L et al. Angiotensin Ⅱ and potassium regulate human CYP11B2 transcription through common cis-elements．Mol Endocrinol，1997，11：638-49．
14．Komiya I，Yamada T，Takara M et al．Lys(173)Arg and-344T/C variants of CYP11B2 in Japanese patients with low-renin hypertension．Hypertension，2000，35：699-703．
15．Zhu H，Sagnella GA，Dong Y et al．Contrasting associations between aldosterone synthase gene polymorphisms and essential hypertension in blacks and in whites．J Hypertens，2003，21：87-95．
16．Barr M，MacKenzie SM，Friel EC et al．Polymorphic variation in the 11beta-hydroxylase gene associates with reduced 11-hydroxylase efficiency．Hypertension，2007，49：113-9．
17．Imrie H，Freel M，Mayosi BM et al．Association between aldosterone production and variation in the 11beta-hydroxylase (CYP11B1) gene．J Clin Endocrinol Metab，2006，91：5051-6．
18．Mulatero P，Schiavone D，Fallo F et al．CYP11B2 gene polymorphisms in idiopathic hyperaldosteronism．Hypertension，2000，35：694-8．
19．龚艳春，张华，初少莉，等．原发性醛固酮增多症亚型临床及基因分型研究．上海医学，2007，30：51．
20．Conn JW．Presidential address．Ⅰ．Painting background．Ⅱ．Primary aldosteronism，a new clinical syndrome．J Lab Clin Med，1955，45：3-17．
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31．陈丹，王琳，薛雨，等．醛固酮合成酶CYP11B2基因C-344T多态性与原发性高血压相关性研究．大连医科大学学报，2006，28：446-9．
32．牛文全，王建炳，李素洁，等．北方汉族人群醛固酮合成酶基因多态性与高血压的关系．中国医学科学院学报，2007，29 329-335．
33．许骥，华琦，李东宝，等．醛固酮合成酶基因CYP11B2 (-344T/C)多态性对北京汉族人群高血压患者缬沙坦降压疗效的影响．中国实用内科杂志，2007，27：124-6．
34．呼邦传，初少莉，王继光，等．醛固酮合成酶基因-344T/C多态性与高血压患者血浆醛固酮及血管紧张素Ⅱ水平相关．中华医学杂志，2006，86：1144-7．
35．Keavney B，Mayosi B，Gaukrodger N et al．Genetic variation at the locus encompassing 11-beta hydroxylase and aldosterone synthase accounts for heritability in cortisol precursor (11-deoxycortisol) urinary metabolite excretion．J Clin Endocrinol Metab，2005，90：1072-7．
36．Freel EM，Ingram M，Wallace AM et al．Effect of variation in CYP11B1 and CYP11B2 on corticosteroid phenotype and hypothalamic-pituitary-adrenal axis activity．in hypertensive and normotensive subjects．Clin Endocrinol (Oxf)，2008，68：700-6．
37．Ganapathipillai S，Laval G，Hoffmann IS et al．CYP11B2-CYP11B1 haplotypes associated with decreased 11 beta-hydroxylase activity．J Clin Endocrinol Metab，2005， 90：1220-5．
38．Kennon B，Ingram MC，Friel EC et al．Aldosterone synthase gene variation and adrenocortical response to sodium status，angiotensin Ⅱ and ACTH in normal male subjects．Clin Endocrinol (Oxf)，2004，61：174-81．
39．Rao A，Melby JC．Idiopathic hyperplasia of the adrenal gland behaving like an aldosterone producing adenoma．J Endocrinol Invest，1997，20：29-31．
40．Shinbo H，Suzuki K，Sato T et al．Simultaneous bilateral laparoscopic adrenalectomy in ACTH-independent macronodular adrenal hyperplasia．Int J Urol，2001，8：315-8．
41．Lamas C，Alfaro JJ，Lucas T et al．Is unilateral adrenalectomy an alternative treatment for ACTH-independent macronodular adrenal hyperplasia? Long-term follow-up of four cases．Eur J Endocrinol，2002，146：237-40．
42．吴瑜璇，祝宇．特发性醛固酮增多症手术是否必要?中华内分泌代谢杂志，2005，21：475-6． rs1799998, intron 2W/C, rs4539 and rs6387, which were not significantly associated with PA , may be potentially relevant to PA. All the susceptible haplotypes included allele A, which confirmed the relationship between the rs 6410 and PA. However, there are also some limitations in our study. First, the patients with APA and IHA were recruited from the inpatients subjected to unilateral adrenalectomy, which was not completely free from some bias in selecting the study subjects. A further study in typical population is useful to reveal the precise association between the polymorphisms in CYP11B2 and CYP11B1 genes and PA. Second, we could not obtain enough information on some environmental exposure, such as smoking and drinking status, and occupational history in both case and control groups, which may bias our results. Third, larger samples from normal population and patients with APA and IHA will be needed in the further study. In summary, our results reveal highly significant association between genetic variations in CYP11B2 and CYP11B1 genes and genetic predisposition to PA. Although we cannot conclude with confidence that rs6410 variant is a causal polymorphism for PA, the significant global and haplotype specific P values support the potential role of CYP11B2 and CYP11B1 genes in the genetic etiology of PA. Additional research, especially with more polymorphisms, typical objects, more environmental exposure, and other study designs, is warranted to explore further the etiologic relevance of polymorphisms in CYP11B2 and CYP11B1 genes for APA and IHA.
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29．陈丹，王琳，薛雨，等．醛固酮合成酶CYP11B2基因C-344T多态性与原发性高血压相关性研究．大连医科大学学报，2006，28：446-9．
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32．Barr M，MacKenzie SM，Friel EC et al．Polymorphic variation in the 11beta-hydroxylase gene associates with reduced 11-hydroxylase efficiency．Hypertension，2007，49：113-9．
33．Keavney B，Mayosi B，Gaukrodger N et al．Genetic variation at the locus encompassing 11-beta hydroxylase and aldosterone synthase accounts for heritability in cortisol precursor (11-deoxycortisol) urinary metabolite excretion．J Clin Endocrinol Metab，2005，90：1072-7．
34．Freel EM，Ingram M，Wallace AM et al．Effect of variation in CYP11B1 and CYP11B2 on corticosteroid phenotype and hypothalamic-pituitary-adrenal axis activity in hypertensive and normotensive subjects．Clin Endocrinol (Oxf)，2008，68：700-6．
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36. Ganapathipillai S, Laval G, Hoffmann IS et al. CYP11B2-CYP11B1 haplotypes associated with decreased 11 beta-hydroxylase activity. J Clin Endocrinol Metab, 2005,90:1220-5.
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53. Fardella CE, Mosso L, Gomez-Sanchez C et al. Primary hyperaldosteronism in essential hypertensives: prevalence, biochemical profile, and molecular biology. J Clin Endocrinol Metab, 2000, 85:1863-7.
54. Lim PO, Dow E, Brennan G et al. High prevalence of primary aldosteronism in the Tayside hypertension clinic population. J Hum Hypertens, 2000,14:311-5.
55. Mosso L, Carvajal C, Gonzalez A et al. Primary aldosteronism and hypertensive disease. Hypertension, 2003, 42:161-5.
56. Rossi GP, Bernini G, Caliumi C et al. A prospective study of the prevalence of primary aldosteronism in 1,125 hypertensive patients. J Am Coll Cardiol, 2006,48:2293-300.
57. Schwartz GL, Turner ST. Screening for primary aldosterbnism in essential hypertension: diagnostic accuracy of the ratio of plasma aldosterone concentration to plasma renin activity. Clin Chem, 2005,51:386-94.
58. Freel EM, Connell JM. Mechanisms of hypertension: the expanding role of aldosterone. J Am Soc Nephrol, 2004,15 :1993-2001.
59. Inglis GC, Plouin PF, Friel EC et al. Polymorphic differences from normal in the aldosterone synthase gene (CYP11B2) in patients with primary hyperaldosteronism and adrenal tumour (Conn's syndrome). Clin Endocrinol (Oxf), 2001, 54:725-30.
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61．Tanahashi H，Mune T，Takahashi Y et al．Association of Lys173Arg polymorphism with CYP11B2 expression in normal adrenal glands and aldosterone-producing adenomas．J Clin Endocrinol Metab，2005，90：6226-31．
62．龚艳春，张华，初少莉，等原发性醛固酮增多症亚型临床及基因分型研究．上海医学，2007，30：51．
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13. Streeten DH, Tomycz N, Anderson GH. Reliability of screening methods for the diagnosis of primary aldosteronism. Am J Med, 1979, 67:403-13.
14. Fardella CE, Mosso L, Gomez-Sanchez C et al. Primary hyperaldosteronism in essential hypertensives: prevalence, biochemical profile, and molecular biology. J Clin Endocrinol Metab, 2000, 85:1863-7.
15. Lim PO, Dow E, Brennan G et al. High prevalence of primary aldosteronism in the Tayside hypertension clinic population. J Hum Hypertens, 2000,14:311-5.
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22. Barbato A, Russo P, Siani A et al. Aldosterone synthase gene (CYP11B2) C-344T polymorphism, plasma aldosterone, renin activity and blood pressure in a multi-ethnic population. J Hypertens, 2004, 22:1895-901.
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42．吴瑜璇，祝宇．特发性醛固酮增多症手术是否必要?中华内分泌代谢杂志，2005，21：475-6． rs1799998, intron 2W/C, rs4539 and rs6387, which were not significantly associated with PA , may be potentially relevant to PA. All the susceptible haplotypes included allele A, which confirmed the relationship between the rs 6410 and PA. However, there are also some limitations in our study. First, the patients with APA and IHA were recruited from the inpatients subjected to unilateral adrenalectomy, which was not completely free from some bias in selecting the study subjects. A further study in typical population is useful to reveal the precise association between the polymorphisms in CYP11B2 and CYP11B1 genes and PA. Second, we could not obtain enough information on some environmental exposure, such as smoking and drinking status, and occupational history in both case and control groups, which may bias our results. Third, larger samples from normal population and patients with APA and IHA will be needed in the further study. In summary, our results reveal highly significant association between genetic variations in CYP11B2 and CYP11B1 genes and genetic predisposition to PA. Although we cannot conclude with confidence that rs6410 variant is a causal polymorphism for PA, the significant global and haplotype specific P values support the potential role of CYP11B2 and CYP11B1 genes in the genetic etiology of PA. Additional research, especially with more polymorphisms, typical objects, more environmental exposure, and other study designs, is warranted to explore further the etiologic relevance of polymorphisms in CYP11B2 and CYP11B1 genes for APA and IHA.
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