| IntroductionPrimary 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. Methods1.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.Results1.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 (χ2=7.39,P=0.007;χ2=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).Conclusions1.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... |
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