Genetic and functional analyses of aldosterone synthase gene C-344T polymorphism with essential hypertension

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Hypertension is a polygenic disorder with a train of genes listed as potential candidates. Thereof, the aldosterone synthase gene ( CYP11B2 ) ranks high on account of its pivotal role in catalysing the biosynthesis of aldosterone. 1 The overall effect of aldosterone is to increase re-absorption of ions and water in the kidney, and thus lead to elevated blood pressure.

Widely evaluated within CYP11B2 concerns a promoter polymorphism, C-344T (rs1799998), which is located in an enhancer element that affects a putative steroidogenic factor-1 (SF-1) binding capacity.2 So far, dozens of studies associating C-344T with hypertension have been conducted, whereas results are often irreproducible.3, 4, 5, 6 It is generally accepted that an association study, coupled with functional validation, represents a powerful approach to deciphering the genetic makeup of complex disease.7 However, a literature search did not trace any functional hints about the promoter activity changes driven by C-344T. Therefore, we aimed to investigate whether the C-344T polymorphism was associated with essential hypertension among the Shanghai Chinese, and to further explore the in vitro biological relevance of this polymorphism to the CYP11B2 promoter activity in 293T cell line, a type of human embryonic kidney line.

This was a hospital-based case–control study involving 950 unrelated Han Chinese in the Shanghai area. There were 475 patients diagnosed with essential hypertension through extensive clinical examinations at the Ruijin Hospital and 475 age- and gender-matched controls (see Supplementary Table 1 for detailed characteristics). The ethics committee of the Ruijin Hospital approved this study protocol, and all subjects gave informed written consent.

Blood pressure (BP) was measured on three occasions with at least 5-min intervals by certified examiners and three consecutive readings were averaged for analysis. Hypertension was defined as systolic BP exceeding 140 mm Hg, or diastolic BP exceeding 90 mm Hg or receipt of antihypertensive medication.

C-344T genotypes were determined by PCR and restriction fragment length polymorphism techniques according to our recent report.8 Genotyping was performed by Niu et al. blinded to the case–control status.

To assess CYP11B2 promoter activity, promoter fragments harbouring the polymorphism −344s were first cloned into the pMD18-T simple vector (TaKaRa, Dalian, China) with forward primer: 5′-IndexTermGATACTCGAGCAGGAGGAGACCCCAT-3′ and reverse primer: 5′-IndexTermGATAAAGCTTCCTCCACCCTGTTCAG-3′. The accuracy of constructs was confirmed by direct sequencing. The fragment incorporated into the CYP11B2 promoter was extracted by restriction enzymes XhoI and HindIII, and then inserted into the multiple cloning sites (XhoI and HindIII) of the pGL3-basic vector. The pGL3 vector contains the cDNA encoding firefly luciferase. The vector containing the −344CC genotype was designated as pGL3-C and the vector containing the −344TT genotype as pGL3-T. The insert orientation in the CYP11B2-pGL3 construct was confirmed by sequencing. HEK293 cells cultivated in minimal essential medium containing 10% fetal calf serum were plated into 6-well dishes (5 × 105 per well), grown for 24 h and transfected using Lipofectamine 2000 (Invitrogen, Shanghai, China). Finally, 4.0 μg of the pGL3 vector and 1.0 μg of the pRL-TK vector (encoding the Renilla luciferase used as an internal control) was co-transfected into cells, which were grown for 36 h. Cell lysates were prepared, and luciferase activity was measured with a luminometer using a dual luciferase assay system. Each promoter activity was directly measured by the ratio of the firefly luciferase level to the Renilla luciferase level. For each reporter construct, five independent transfection experiments were conducted.

Statistical calculations were performed using the R software (http://www.r-project.org). The χ2 test was used to assess the goodness of fit of the Hardy–Weinberg equilibrium and differences in genotype/allele distributions between case–control groups. Student's t-test was applied to compare the luciferase assay data. Statistical significance was set at two-tailed P<0.05.

With regard to C-344T association (Figure 1a), no deviation from the Hardy–Weinberg equilibrium was observed and no statistical differences in the genotype and allele distributions existed between cases and controls (P>0.05). As shown in Figure 1b, the pGL3 empty vector had a negligible amount of luciferase activity. The CYP11B2 promoter harbouring −344C conferred lower transcription activity compared with its counterpart, while no statistical significance was reached (P=0.4444).

Figure 1
figure1

Distribution of C-344T genotype/alleles between cases and controls (a) and comparison of the promoter activity of its two alleles in CYP11B2 (b). In panel a, the black square represents the major homozygous genotype (−344TT) or major allele (−344CC), the grey square heterozygous genotype (−344CT), and the white square minor homozygous genotype or minor allele. The numbers in squares denote genotype counts or allele frequencies. In panel b, the promoter activity was examined using a luciferase reporter system in human embryonic kidney line, 293T. Values represent the average of five experiments and the bars represent s.d. The pGL3-basic was used as a negative control without any promoter sequence, and pGL3-control as a positive control.

Exceeding our expectation, the C-344T polymorphism was not associated with essential hypertension among the Shanghai Chinese, and no significant difference was noted between the transcriptional activity of the CYP11B2 promoter harbouring −344C and −344T alleles as well. Therefore, the concordance of results between association and function has substantiated the irrelevance of C-344T to hypertension. To the authors’ knowledge, this study is the first to explore the biological relevance of the C-344T polymorphism to CYP11B2 promoter activity in 293T cell line.

Although great efforts have been taken to explore the genetic susceptibility of C-344T to hypertension, a considerable degree of non-reproducibility tingled most of these studies.3, 4, 5, 6 On the basis of a recent meta-summarization on 42 association studies, Sookoian et al.9 suggested that −344CC homozygotes had a lower risk of hypertension than did their −344TT counterparts, whereas no significance held for the overall risk estimates from the random-effects model considering the clear between-studies heterogeneity. Further pooled phenotype–genotype association on plasma aldosterone levels as well as systolic and diastolic arterial BP still yielded no positive evidence. Although the authors have emphasized the cross-ethnic discrepancy in these associations,9 most of their studies enrolled have produced negative results, especially in Asians. Similarly, negative findings regarding the association of C-344T with essential hypertension was also observed in our recent report from Northern Chinese (the Fangshan Study)8 and from the present association study in Southern Chinese. Therefore, it is nothing to be surprised at the negative relevance of CYP11B2 C-344T polymorphism to its promoter activity in our experiment.

Functional evidence indicated that C-344T is located in the binding site of SF-1, which can downregulate the promoter activity. As estimated, the in vitro affinity of the −344T allele for SF-1 was five times lower than that of the −344C allele.2 This was consistent with the higher, although non-significant, level of transcriptional activity driven by the −344T allele than the −344C allele. However, experimental data disfavoured the positive regulation of SF-1 on CYP11B2.2 It thus leaves open the possibility that C-344T itself does not appear to be functional. Considering the fact that results from experiments in vitro may not reflect the regulation of gene transcription in vivo, the jury must refrain from drawing a firm conclusion until further verification of our finding in vivo.

Taken together, this external and internal consistency from the genetic and biological results stands as proof of the irrelevance of CYP11B2 C-344T polymorphism to the development of hypertension.

References

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Acknowledgements

This work was financially supported by the Shanghai “Chen Guang” Project (CG0912), Natural Science Foundation of Shanghai (09ZR1426200 and 08ZR1422000), two items of Excellent Young Teachers Program from Ruijin Hospital and Shanghai City (JDY08025), Science Fund of Shanghai Jiaotong University School of Medicine (09XJ21019), Shanghai Education Committee (09YZ101) and the National Science Foundation for Young Scientists of China (Grant no.: 30900808).

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Correspondence to W-Q Niu or S-J Guo.

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The authors declare no conflict of interest.

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Supplementary Information accompanies the paper on the Journal of Human Hypertension website

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Niu, W., Guo, S., Zhang, Y. et al. Genetic and functional analyses of aldosterone synthase gene C-344T polymorphism with essential hypertension. J Hum Hypertens 24, 427–429 (2010) doi:10.1038/jhh.2010.12

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