Association Between Essential Hypertension And Polymorphism Of The SLC12A3 Gene In Clustered Mongolian Populations

Objective: This study took Mongols population in Mongols inhabited areas as the research object,to explore the relationship between SLC12A3 gene polymorphism and its expression level and Mongols essential hypertension,and Functional verification was carried out on the newly discovered Mongols hypertension susceptibility site SLC12A3 rs5805 in this study,providing a new target for the development of hypertension drugs caused by this type of mutation.Method:(1)The research population of this study mainly comes from the Mongols hypertension case-control population collected in Mongols Autonomous Region(Zhenglan Banner,Xilinhot City,Dongwu Banner,Xianghuang Banner)from August 2017 to September2019,with a total of 335 people,including 235 hypertension patients and 100 normal controls.(2)This study screened SNPs sites on the SLC12A3 gene by searching the NCBI database and consulting literature.Multiple high-temperature ligase reaction technology was used for genotyping of SNPs,Epidata3.0 software was used to collect and sort out data,and SNPStats software SNPStats: your web tool for SNP analysis.The association analysis of SNPs alleles,genotypes and essential hypertension was carried out.The genetic models analyzed included Codominance model,dominant model,recessive model,Overdominance model and additive model.(3)Ultrahigh speed centrifugation of the urine of the subjects,nano particle tracking analysis(NTA)technology,transmission electron microscopy confirmed that the centrifugation product was an extra urinary body.3DLabel Free proteome sequencing was performed in a case control population consisting of 77 Mongols hypertensive patients and 33 normal blood pressure subjects.(4)The SLC12A3 rs5805 wild type and mutant plasmids were synthesized and transfected into 293 T cells.The double Luciferase reporter gene experiment was used to detect whether SLC12A3 rs5805 mutation affected the expression of NCC protein,and the fluorescence intensity of 293 T cells after 48 hours and 72 hours of transfection were detected respectively.Conduct three independent experiments and calculate the mean,and compare the differences between the two groups using SPSS 25.0 software.Apply G-power software for statistical efficacy analysis.Result:(1)Six SNPs(rs2304479,rs5801,rs5802,rs5804,rs2289113,and rs5805)were ultimately included in this study,all of which met the Hardy Weinberg equilibrium(P > 0.05),proving that the study subjects we selected were random populations and could be used for subsequent association analysis;(2)The results of SNPStats software association analysis showed that the GG genotype of rs5805 was significantly associated with Mongols primary hypertension.The Codominance model(OR=2.38,95% CI=1.15-4.96,P = 0.04),the recessive model(OR=2.13,95% CI=1.08-4.17,P = 0.03)and the additive model(OR=1.46,95%CI=1.03-2.08,P = 0.03)suggested that individuals carrying the GG genotype had a higher risk of hypertension;However,in the hypertension group and the control group,other SNPs alleles and genotypes were not found to be associated with Mongols essential hypertension.(3)Centrifuge extracted urine samples were confirmed as exosomes,and sequencing results showed that 88 proteins were upregulated in the hypertension group,including the SLC12A3 gene encoding protein NCC.This difference was only observed in the overall population(EH/NC ratio=1.724,P = 0.01)and female population(F-EH /F-NC ratio=1.968,P = 0.04);(4)The rs5805 wild-type and mutant plasmids were synthesized and transfected into 293 T cells,respectively.After 48 and 72 hours,the fluorescence intensity of the two groups was detected.The results showed a statistically significant difference in fluorescence intensity between the two groups(P < 0.05),and the fluorescence intensity of the mutant group was higher than that of the wild-type group.Conclusion:(1)The rs5805 polymorphism of SLC12A3 gene is associated with the risk of essential hypertension in Mongols population in Mongols inhabited areas;(2)The expression of NCC in the urinary tract of hypertensive patients in Mongols population was significantly higher than that in the control group;(3)The mutation of rs5805 site will affect the expression of NCC.