A Study On The Association Between CYP7A1 Gene Rs1125226 Polymorphism And Essential Hypertension

PrefacePrevalence of Essential hypertension accounted for more than 95% of hypertension. As one of the most important risk factors for cardiovascular disease, more and more attentions had been paid for it. In recent years, the prevalence of EH is increasing year by year. EH has become a major public health problem impacting the population health. EH is a polygenic disease affected by environmental factors and genetic factors. Because of genetic factors in the pathogenesis of EH playing an important role, looking for single-nucleotide polymorphisms (SNPs) of its associated genes and then clarifying the genetic mechanism of EH disease has become the research hotspot currently.We carried out a population survey of a large sample in the western region of Liaoning Province since 2001. We found that the standardized prevalence rate of hypertension was as high as 35%. Such a high prevalence is rare in the country. We also found that about 50% patients with hypertension had a high level of triglyceride and low-density lipoprotein (LDL). This suggests that people in the area of high incidence of EH may be associated with an imbalance of lipid metabolism. Existing research data shows that the occurrence and development of EH is closely related to dyslipidemia. But it have been inconclusive whether the two share a common genetic basis.Cholesterol 7a-hydroxylase gene (CYP7A1 gene) is the important gene in lipid metabolism. This study found a single-nucleotide polymorphisms rs1125226 in the upstream regulatory region of CYP7A1 gene through the SNPs database of NCBI. There is no report on whether the polymorphism of rs1125226 is associated with the occurrence of EH. Therefore, this study is intended to examine whether the rs1125226 polymorphism of CYP7A1 gene is associated with the occurrence of EH and its associated degree through case-control study.Subjects and Methods1,The selection of subjectsAll of the subjects were selected from 6000 population in Zhangwu County, Liaoning Province. All the people from 25 to 65 years old were investigated.300 cases with hypertension were selected as case group, and secondary hypertension was excluded.300 subjects without hypertension were selected from the same population.2,Diagnosis standard and the methods of measurementsThe diagnosis standard of EH:According to 1999 WHO-ISH guidelines for the management of hypertension, hypertension was defined as a systolic blood pressure (SBP)≥140mmHg and/or a diastolic blood pressure (DBP)≥90mmHg. The grading standard of EH:the first grade:140mmHg≤SBP<160mmHg and/or 90mmHg≤DBP <100mmHg; the second grade:160mmHg≤SBP<180mmHg and/or 100mmHg≤DBP<110mmHg; the third grade:SBP≥180mmHg and/or DBP≥110mmHg. The measurement of blood pressure should be carried out according to the unified standard under standard conditions.3,The contents of epidemiological surveyQuestionnaires were filled by means of inquiring and measurement in sites. The contents of survey included:①General characteristics:including age, gender, nationality, home address, family size, etc.②Hypertension-related factors:including alcohol intake, smoking habits, family history of hypertension and other disease history and so on.③Physical examination:measuring of the blood pressure, body height and weight calculating body mass index.④Blood samples:five milliliters blood samples were drawn after an overnight fast. After centrifugation, the serum fraction was removed. Then, six milliliters blood samples were drawn without centrifugation. All the blood samples were frozen at-20℃until assayed. 4,The measurement methods of the indicatorsTotal cholesterol (TC), triglyceride (TG), high-density lipoprotein (HDL), low-density lipoprotein (LDL), serum sodium, serum potassium, serum iron, serum calcium were measured by automatic biochemistry analyzer 7150 (HITACHI, Japan). The blood sugar was measured by blood sugar analyzer (Johnson & Johnson, America).The CYP7A1 gene rs1125226C/A SNP was detected by Real-time PCR (ABI 7500). The Taqman-MGB probes and primers were provided by Applied Biosystems.5,Statistical methodsA database was established by SPSS13.0. Quantitative data were expressed as mean±standard deviation (X±SD) and compared the difference between the two groups by the independent-samples t test. Qualitative data and Hardy-Weinberg equilibrium were tested usingχ2 test. Multivariate analysis was using multiple stepwise Logistic regression method. Statistical significant level defined asα=0.05.Results1,Analysis for equitability between the case group and control groupThere was no statistical difference of the distribution on sex and age between case group and control group. The indicators of systolic blood pressure, diastolic blood pressure, proportion of alcohol, BMI, cholesterol, triglyceride, low-density lipoprotein, serum sodium, serum iron and serum calcium in case group were higher than in control group; indicators of high-density lipoprotein and serum potassium in case group were lower than in control group. The difference was statistically significant (P<0.05).2,The distribution of CYP7A1 gene rs1125226 polymorphism between the case group and control group(1) The Hardy-Weinberg equilibrium test and allele frequency in the study population The genotype frequency and allele frequency distribution were consistent with Hardy-Weinberg equilibrium(χ2=3.752, P=0.053) in this study. The frequency of allele A in our study population was 0.3817. It was consistent with the frequency of allele A (0.38) that HapMap reported in Han Chinese population.(2) The distribution of genotypes and allele between the case group and control groupThere was no statistical difference on frequency of genotypes and allele of CYP7A1 gene rs1125226 SNP between case group and control group after correlation analysis.(3) The distribution of genotypes and allele in patients with hypertensionIn order to explore the distribution of the genotype and allele in the patients with EH, patients with hypertension in our study were divided into 3 groups separately in accordance with the levels of high blood pressure, systolic and diastolic blood pressure. There was also no statistical difference on frequency of genotypes and allele of CYP7A1 gene rs1125226 SNP in patients with varying degrees of hypertension.(4) The distribution of genotypes and allele between the case group and control group in different gender groupThere was no statistical difference of the distribution of genotypes and allele in different gender group between case group and control group after that the case-control study was stratified by sex factor.(5) The distribution of genotypes and allele between the case group and control group in different age groupThe distribution of genotype frequency between the EH group and control group emerged statistically significant difference in the 25-35 years age group and 35-45 years age group after that the case-control study was stratified by age factor (χ2=7.31, P=0.026 andχ2=6.66, P=0.036). AA genotype frequency (25%) in the EH group was higher than the control group (20%) in the 25-35 years age group; and AA genotype frequency (26.6%) in the EH group was much higher than the control group (10.8%) in the 35-45 years age group. 3,Multi-factor analysis for hypertension in study populationLogistic regression analysis showed that:the genotype of rs1125226 SNP as independent variable failed to enter the model of high blood pressure after adjusted by other factors. The indicators of alcohol intake, BMI, triglyceride, low-density lipoprotein, serum sodium and serum potassium as independent variables entered the model. These indicators were risk factors for hypertension except serum potassium(adjusted OR=0.514,95%CI:0.319～0.827).DiscussionThere are 146 patients with abnormal level of TC (49.2%),96 patients with abnormal level of TG (32.3%),109 patients with abnormal level of LDL (36.7%) and 21 patients with abnormal level of HDL (7.1%) in 298 patients with EH included in this study. And 193 people (64.98%) at least have one of four indicators outside the normal range. It can be seen that population with EH in this study combined dyslipidemia is very common, which is similar to other research findings. It indicated that the occurrence of EH in this region may be closely related to dyslipidemia, and the existence of common genetic basis in blood pressure regulation and lipid metabolism.Up to date, there are at least 150 candidate genes of EH and their corresponding polymorphisms sites identified by linkage analysis and candidate gene association analysis. In fact, some different genetic polymorphisms are associated with a certain race or people-related. These facts can clarify the complexity of EH. There may be the combined effects from multiple "micro-effect genes" in different groups of people with EH. Sometimes a single gene has the limited influence for blood pressure but the combined effects from some minor genes may influence blood pressure by reaching a threshold and promote the pathogenesis of EH. This could be the reason that our study did not find statistical correlation between CYP7A1 gene and the occurrence of EH in the population from the western region of Liaoning. However, statistical correlation was found in middle-aged people (25-45 years), and AA genotype may be an independent risk factor for EH in this middle-aged people (adjusted OR=3.399, 95%CI:1.299～8.893,P=0.013).In addition, this study analyzed the genetic and environmental factors of EH by the model of multiple Logistic regressions. Results showed that:the genotype of rs1125226 SNP as independent variable failed to enter the model. We also found that high levels of LDL and drinking as risk factors for EH with OR> 1.5 did come from the bad habits for local people. Therefore, the high incidence of EH in the region is likely to be controlled if the residents of the region can improve the living habits of these two aspects.ConclusionsCYP7A1 gene rs1125226 polymorphisms may not be related with the occurrence of EH in Zhangwu population in Liaoning Province. However, it may be related to the occurrence of EH in middle-aged people (25-45 years) in which AA genotype may be an independent risk factor for EH. In addition, multi-factor analysis showed that: drinking, high levels of BMI, TG, LDL, serum Na and low level of serum K were the independent risk factors for EH in the region of Zhangwu.