| 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-△△CT 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.
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