| 1. IntroductionThe causes of essential hypertension consist of heredity, environment,habits and dietary history. All of these factors contribute to thedevelopment of hypertension interactively. Although some hypertensionrelated factors have been generally accepted by the scientists, the othersare still controversy. For the existence of genetic instability, especiallythe intervention of foreign nationality genes, it is a challenge to isolatethe genetic influence related to the development of essentialhypertension. Mongolians, the ninth most common minority nationalitypopulation of China, generally lived in a relatively isolated community, and some of them follow the old nomadic traditions, they seldommarries with other nationalities, which make them better candidates forgenetic study.According to the latest report of National Blood Pressure SurveyCooperative Group of China in, the prevalence of hypertension inMongolians (20.2%) is significantly higher than that in Han populationand the total prevalence in Chinese. Habits, living style and heredity areall important factors that associated with the prevalence of hypertensionin different nationalities. Many association studies had been done in Hanpopulation to figure out risk factors for hypertension in recent years.However, little data is available for the reason of high prevalence ofhypertension in Mongolians.From October2010to July2012, a case-control study was organizedby the Inner Mongolia International Mongolian Traditional Medicine Hospital with the cooperation of First Affiliated Hospital of InnerMongolia Medical Colledge, and the Second Affiliated Hospital ofJilin University, aimed to determine the factors that associated with thedevelopment of essential hypertension in Mongolians, and to collecttimely and sufficient information to support future hypertensionprevention program in Mongolian population.2. Methods2.1Study populationStudy sujects were enrolled from Dongwu County, Xilin Gol League,Inner Mongolia Autonomous Region, China. A hypertensive subject whomet the following criteria were enrolled:(a) was descendant ofMongolian;(b) was aged between35and65years;(c) was a permanentresident of local area;(d) had systolic blood pressure (SBP)≥140mmHg and/or diastolic blood pressure (DBP)≥90mmHg;(e) was not taking any antihypertensive medications within1month before the study;(f) was not secondary hypertension and was not presently pregnant; and(g) was willing to participate in the present study. The controls shouldmeet the criteria a, b, c, f and g mentioned above, and their SBP andDBP should lower than140and90mmHg, respectively.2.2Study designEligible subjects (including controls) were invited for a face-to-faceinterview, and a socio-demographic characteristics based questionnairewas designed to collect information on lifestyle, habits and dietaryhistory. Written informed consent was obtained by signature orthumbprint. After the interview, all subjects underwent a blood pressurecheck in local hospitals, venous blood samples were collected for bloodbiochemical examination and human tissue kallikrein1(KLK1) genepolymorphisms analysis. All examinations were provided at no cost to the participant, and all studies were approved by the Ethics Committeeof the First Affiliated Hospital of Inner Mongolia Medical Colledge.2.3Measurement of blood pressure and anthropometricsBlood pressure were measured by well-trained nurses using amercury-gravity manometer with appropriate sized cuffs after thesubjects kept silent in a seated pose for30minutes. Triplicatemeasurements were carried out on the left arm with a one minuteinterval between each reading. We defined SBP and DBP as Korotkoffphase I and V, respectively. The mean of the three blood measurementswas used in statistical analysis.Height, waistline, hipline and weight were used to evaluate thesomatotype of study subjects. Triplicate measurements were carried outby3different interviewers, and the mean value was used in statisticalanalysis. 2.4Blood biochemical examinationBlood samples were collected after an overnight fast, and total plasmacholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol(HDL-C) and low-density lipoprotein cholesterol (LDL-C) weremeasured within eight hours in a local hospital.2.5DNA extraction and KLK1gene polymorphisms analysisGenomic DNA was extracted from peripheral blood leukocytes usinga commercial blood DNA extraction kit and was stored at-20℃. Thesix tagSNPs of KLK1were genotyped using the PCR/ligase detectionreaction assay. Primers were synthesized by Shanghai Sangon BiologicalEngineering Technology&Services. Each set of ligase detectionreaction probes comprised one common probe and two discriminatingprobes.The target DNA sequences were amplified using a multiplex PCR method. PCRs for each subject were carried out in a final volume of20μL, containing1×PCR buffer,3.0mM MgCl2,2.0mM deoxynucleotidetriphosphates,0.2pM primers,0.3μL Qiagen HotStarTaq Polymerase,4μL of1×Q-solution, and10-20ng genomic DNA. Thermal cycling wasperformed in Gene Amp PCR system9600with an initial denaturationof2min at95℃, followed by35cycles of denaturation at94℃for30s,annealing at54℃for1.5min, and extension at72℃for1min,followed by a final extension at72℃for10min.The ligation reaction for each subject was carried out in a finalvolume of10μL, containing1×NEB Taq DNA ligase buffer,0.05pM ofeach probe mix,0.05μL Taq DNA ligase, and1μL of multi-PCR product.The ligase detection reaction was performed with95℃for2min,followed by30cycles of94℃for30s, and50℃for25s. Thefluorescent products of ligase detection reaction were differentiated by 5%polyacrylamide gel electrophoresis and data were collected byGENESCANTM627software.2.6Statistical analysisThe Student’s t-test was used to evaluate the body mass index (BMI),waistline, hipline, as well as blood biochemical data betweenhypertension patients and controls.The intakes of each kind of food were calculated by the reviewer basedon the description of the interviewee during the interview. The intakeswere divided into certain ranks according to the mean consumption oflocal area. Generally, the status of the recent10years in the dietaryhistory was taken into account. Rank sum test was used to evaluate theintakes between hypertensives and controls, and to assess the cigaretteand alcohol intake as well as the education between two groups.Hardy–Weinberg equilibrium (HWE) of KLK1genotype distribution in hypertensives and controls was tested by HWE program. The chi-squaretest was used to compare the allele and genotype frequencies betweenhypertensives and controls. The chi-square test was also used to comparethe proportion of high salt diet population, physical exercisers, andmental laborers/manual workers between two groups.All of data was analyzed by SPSS version16software. Statisticalsignificance was accepted if the P≤0.05. All P values were two-sided.3. Results3.1Study populationTotally194hypertensives and201controls were enrolled in the study.The mean age of two groups was47.51±10.31and48.13±9.85, and theratio of male to female was93/101and92/109, respectively. There wasnot statistically significant difference in age (P=0.937) and the ratio ofmale to female (P=0.913) between two groups. The mean SBP and DBP of hypertensives were165.4±27.1and102.2±16.1mmHg, which wassignificantly higher than those of controls (120.4±11.8mmHg for SBPand78.6±7.3mmHg for DBP, P <0.001).Almost all participators were willing to undergo all researches in thisstudy, except4hypertensives and8controls refused to provide venousblood samples, and34hypertensives and21controls refused toparticipate in the polymorphism analysis of KLK1gene for uncertainreasons.The mean BMI, waistline, and hipline in hypertentives were allsignificantly higher than those in controls (P <0.001for all). In fact, wefound that the “pear” shaped body was more common observed in thehypertensives during the face-to-face interview.Except HDL, the mean concentration of blood sugar, blood ureanitrogen, creatinine, uric acid, total cholesterol, triglyceride and low-density lipoprotein in the venous blood samples from hypertensiveswere all significantly higher than those in controls (P <0.01for bloodsugar and BUN, P <0.001for CRE, UA, TCHO, TG and LDL, Table2).3.2Dietary history, life style, habit, education, occupation andinheritance.Nearly7percent data was excluded in the dietary history analysis. Themain reason was the participators could not describe the daily dietaryclearly, or there was a great change in the recent10years dietary historydue to a sudden disease or other uncertain reasons.The milk, vegetable and fruit intakes in hypertensives were allsignificantly less than those in controls (P <0.05for all). However, theintakes of egg, meat, bean and bean products had no difference betweentwo groups (P>0.05for all).Smoking, drinking, high salt diet and physical exercise were also investigated in the study, the status in recent10years were mainly takeninto account. In evaluating high salt diet, we found that the three meals isnot an exclusive way for salt intake, the salty snacks such as jerked beef,dry pickles are popular in local residents. The salt intake from thesesnacks was also taken into account besides daily meals. The consumptionof salt, cigarette and alcohol were calculated by the interviewer accordingto the statement of study subjects.After all data normalized, we found that the consumption of cigaretteand alcohol in hypertensives was all higher than that in controls (P <0.05for cigarette consumption and P <0.001for alcohol consumption), andthe proportion of subjects with high salt diet in hypertentives was higherthan that in controls (P <0.01). However, the proportion of subjects whodid physical exercise regularly in hypertensives was significantly lowerthan that in controls (P <0.01). No significant difference of educations was found between the twogroups (P>0.05). In this study, the occupation was divided into mentallaborer and manual worker, the former mainly included teachers, doctors,policemen and government officers; the latter generally consisted offarmers, herders and industrial workers. We found that the percentage ofmental laborer in hypertensives was significantly higher than that incontrols (P <0.05). In order to eliminate sample bias, the subjects whosemother or father dead before45years old were excluded from theinheritance analysis. For remaining subjects, the mean inheritance rank ofhypertensives was significantly higher than that of controls (P <0.05).3.3KLK1gene polymorphismsWe investigated the association of six SNPs (rs2659058, rs2659103,rs2659104, rs2740502, rs1054713and rs5517) of KLK1gene andhypertension. Genotype frequencies of the six SNPs of KLK1gene satisfied the Hardy-Weinberg equilibrium in subjects (P>0.05). Thedistribution of genotype and allele frequencies of six SNPs showed nodifference between hypertensives and controls (P>0.05).DiscussionDongwu County (E116, N44) locates at north-east of Inner MongoliaAutonomous Region, China, neighboring Mongolia. The permanentresidents of this area are about55thousand. Nearly three quarters ofresidents are Mongolians, and more than40percent of residents areherders. Because most herders lived far away from the downtown whereour survey performed, only one fifth of study subjects who enrolled intothis study were herders.Until nowadays, there is not an epidemiologic screening on theprevalence of essential hypertension in this area. However, several similarstudies performed in Mongolian population may provide some useful information. The prevalence of essential hypertension is37.4%inNaiman County (E120, N43)(Li et al.,2008),34.5%in Hulun BuirLeague (E118, N49),40.2%in Fuxin County (E121, N42), andaccording to a large scale nutrition and health survey in Chinese in2002,the total prevalence of essential hypertension in Mongolians is26.7%,which is significantly higher than that in Han population (22.7%) andtotal prevalence in Chinese (18.8%).Consistent with previous reports, obesity, dietary history, habit,occupation and inheritance are hypertension related risk factors inMongolian population of China. In our opinion, the dietary history is themost important one. Since the development of obesity depends on dietaryhistory to a great extent, and some habits, such as high salt dietary anddrinking which are both tightly associated with dietary history. To oursurprise, the meat intake, a commonly accepted hypertension related factor, is not associated with the development of hypertension inMongolian population. The possible reason for this conflicting result isthat the lower fat contented beef and mutton rather than pork is morepopular in local Mongolian population. And for the first time, we find thatthe milk intake, which is neglected in most association studies, is one ofhypertension related factor in Mongolian population.Although there are still some controversy for the relationship betweensmoking and hypertension, majority of scientists accept that smoking isone of risk factor for the development of hypertension, especially whenthe quantity of cigarette intake are investigated in statistical analysis. Ourresults showed that the blood pressure was influenced by the cigaretteintake.During the investigation we found that the most different lifestylebetween Han and Mongolian population comes from dietary history and habits. A higher meat and salt intakes and lower vegetable and fruitintakes are commonly observed in Mongolians rather than Hanpopulation. In addition, drinking and smoking are more popular inMongolians. For about three quarters of study subjects (male:79.2%,female:71.1%) are drinkers, and more than60percent of study subjects(male:69.1%, female:39.5%) are smokers. Our results suggested that thehigh drinker and smoker proportion, as well as difference in the dietaryhistory are important reasons for the high prevalence of hypertension inMongolian population.In2007, a large-scale KLK1gene polymorphism analysis has beenperformed in Peking, China, the association between the rs5517andessential hypertension was confirmed in a Peking Han population.However, another report from Anhui province of China believed thatrs5516rather than rs5517was associated with hypertension. The conflicting results may come from sampling. Herein, we do not find anyrelationship between hypertension and genetic variations of KLK1genein Mongolian population.In conclusion, our study suggests that dietary history and habits havethe most important influence on the development of essentialhypertension in Mongolian population. Quit smoking and drinking, aswell as change in diet should be considered as the major hypertensionprevention program in Mongolian population. |
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