A Potential Regulatory Single Nucleotide Polymorphism In The Promoter Of The Klotho Gene May Be Associated With Essential Hypertension

BackgroundMice with defects in the Klotho gene exhibit multiple aging phenotypes including a short lifespan, infertility, arteriosclerosis, skin atrophy, osteoporosis and emphysema. Human Klotho, showing 86% amino acid identity with the mouse protein, may be involved in the aging process. In previous studies, more than ten single nucleotide polymorphisms (SNPs) in the human Klotho gene were found to be associated with age-related diseases. Interestingly, two notable changes of Klotho-deficient mice are arteriosclerosis and endothelial dysfunction, which are the fundamental etiological factors of human essential hypertension (EH). However, the association of the Klotho gene SNP with EH in the Chinese population is scarce.ObjectivesIn the present studies, we hypothesized that the G-395A polymorphism in the promoter region of the human Klotho gene may contribute to the prevalence of EH and the G-395A site may be a potential regulatory SNP (rSNP) of the Klotho gene.MethodsThe case-control study was performed in the Chongqing Han population consisting of 215 patients with EH and 220 non-hypertensive subjects. The genotypes of the Klotho gene G-395A SNP were determined by the TaqMan allelic discrimination assay. The distributions of genotype frequencies were analyzed in the two groups and in the subgroups according to age, gender and smoking status. The odds ratios of G-395A SNP and traditional risk factors of EH were computed by the use of a multiple logistic regression model. In addition, the brachial-ankle pulse wave velocity (baPWV) and serum nitric oxide (NO), as the vascular function markers, were compared between the -395A carriers and non-carriers in 232 subjects of the population. By using serial deletion recombinant plasmids of the promoter, the -975bp~-8bp fragment upstream of the Klotho gene was investigated for responsible promoter region in HEK293 cells. Furthermore, whether a G/A substitution at the G-395A site of Klotho gene affected the transcription level in vitro was also tested through the dual-luciferase reporter assay.Results1. In the study subjects, 288 subjects had the GG genotype, 130 subjects had the GA genotype, and 17 subjects had AA genotype. The allele frequencies were 0.809 for the G allele and 0.191 for the A allele. These were in compliance with the Hardy–Weinberg equilibrium (χ2 = 0.107; P = 0.948).2. Differences in the genotype distributions of the G-395A polymorphism between the EH and non-hypertension groups were statistically significant (P = 0.032). There were differential effects of age, gender and smoking status on the association of the G-395A polymorphism with EH; the G-395A polymorphism was significantly associated with EH in subjects over 60 years old, in females and in nonsmokers (P = 0.039, P = 0.001, and P = 0.010, respectively). A multiple logistic regression analysis indicated that the odds ratio for EH in the–395A allele carriers as compared with the control group was 0.593 after adjusting for current traditional risk factors including advanced age, smoking status, being overweight, diabetes mellitus and dyslipidemia (95% CI = 0.376–0.935; P = 0.024).3. There were no statistical differences of G-395A distributions among the different age groups in the study population.4. The ?395A allele carrier of the G-395A polymorphism of the human Klotho gene was found to have a lower baPWV than the non-carrier (1463.8±297.0 vs. 1572.6±347.1, P=0.014), but the serum NO between the two groups were not statistically significant (11.8±6.6 vs. 11.1±6.7; P=0.448).5. The expression of Klotho in HEK293 cells was confirmed by RT-PCR. The dual-luciferase reporter assay of serial deletion of the Klotho promoter revealed that there was a promoter element in the -506bp～-8bp DNA fragment (containing the G-395A site) upstream of the Klotho gene ATG start codon.6. No significant differences of the luciferase activities were seen after the G/A substitution in the pGL3-Basic recombinant plasmid of the Klotho -506bp～-8bp DNA fragment (0.043±0.09 vs. 0.028±0.012 respectively; P = 0.094). However, the–395A carrier in the pGL3-Enhancer recombinant plasmid had a higher relative luciferase activity as compared with the–395G carrier (0.251±0.084 vs. 0.103±0.013 respectively; P = 0.013).ConclusionsThe G-395A polymorphism of the human Klotho gene is associated with EH in Han population and may be a potential regulatory site by altering the transcriptional level of the Klotho gene.