GENETIC POLYMORPHISMS ASSOCIATED WITH HYPERTENSION IN THE ETHNIC POPULATIONS OF CALABAR AND UYO, NIGERIA BY MARY ESIEN KOOFFREH S.I. 72072 B.Sc Biology Genetics (Calabar), M.Sc. Cell Biology and Genetics (Ibadan). A thesis in the Department of Zoology, Submitted to the Faculty of Science in partial fulfillment of the requirements for the Degree of DOCTOR OF PHILOSOPHY Of the UNIVERSITY OF IBADAN AUGUST 2012 ABSTRACT Hypertension is a public health challenge due to its high prevalence, and is a major risk factor for cardiovascular diseases. Hypertension is a complex disease resulting from an interaction of genes and environmental factors. Inconsistent association between polymorphisms of the renin angiotensin aldosterone, the atrial natriuretic peptide systems and hypertension has been reported among various ethnic groups, but not for the Efiks and Ibibios in south-south Nigeria. This study was designed to determine the frequency of gene polymorphisms of these two systems and their association with hypertension in Calabar and Uyo, Nigeria. A population-based case control design was used. A total of 1224 participants, 612 each of patients and controls were randomly recruited from hypertension clinics and the general population. Genotyping of the M235T allele of the angiotensinogen, Insertion/Deletion allele (I/D) of the angiotensinogen converting enzyme, A1166C allele of the angiotensin II type I receptor and C664G allele of the atrial natriuretic peptide genes to identify variants was performed using polymerase chain reaction and restriction enzyme digestion. The Hardy-Weinberg equation was used to calculate the allele and genotype frequencies. Plasma angiotensinogen levels were measured by Enzyme Linked Immunosorbent Assay. Hypertensinogenic factors such as age, familial history, physical exercise and drinking were assessed using questionnaires. Descriptive statistics, chi- square, multiple regression analysis and odds ratio were used to analyze the data. The frequency of the genotypes M235M, M235T, T235T of the M235T allele for the Efiks were 0.4, 7.7, 92 % in patients and 0, 6, 94 % in controls; for the Ibibios were 0.5, 1.2, 87 % in patients and 0, 7, 93 % in controls. The I/D genotypes II, ID, DD frequencies for the Efiks were 11, 44, 46 % in patients and 16, 45, 39 % in controls; for the Ibibios were 11, 40, 49 % in patients and 13, 49, 38 % in controls. The frequency of the A1166C carriers was 1 % while 99 % of the study population had the wild type A1166A genotype for the A1166C allele. Only the CC genotype was observed for the C664G allele. These frequencies did not conform to the Hardy-Weinberg assumptions. There were no significant differences between the genotype frequencies of patients and controls. Plasma ii angiotensinogen values were significantly higher in the patients with M235T allele than in the controls. Age was a positive predictor for systolic blood pressure (SBP, r = 0.60) in patients and diastolic blood pressure (DBP, r = 0.56) in controls. Other hypertensinogenic variables were not predictors for SBP and DBP in the population (p < 0.05). The Insertion/Deletion allele was a risk factor for hypertension, (O.R = 1.15). A high frequency was observed for the M235T allele and the Insertion/Deletion allele, which was associated with an increased risk for hypertension. The lack of association between the alleles of the M235T, A1166C and the C664G and hypertension suggests that other loci or environmental factors are involved in the disease outcome. Keywords: Polymorphism, Hypertension, Allele and genotype, Efiks and Ibibios Word count: 484 iii ACKNOWLEDGEMENTS 1 am deeply grateful to my supervisor Dr C.I. Anumudu for accepting me as her student and her patience in going through this work. Her useful criticisms and advice have made this work successful. I am indebted to Dr Lava Kumar for giving me the opportunity to learn the techniques and carry out the research in his laboratory. I am also grateful to Mr. Segun Akinbade for putting me though the different methods. I will not forget Dr Kamal Sharma, Aunty Pat Ogunsaya, Dr Oby Eni, Mrs. Josephine Ezeri, Mrs. Ronke Oludare, Mr. Deji Owase, Mr. Mark Time, Mr. Taiwo, Mr. Jimoh, Mr. Salaudeen, all of the virology unit of the International Institute for Tropical Agriculture, Ibadan for their contributions, I really appreciate them. My gratitude also goes to Dr E.E. Ephraim, Prof A. Asindi, Dr Akpan, Dr Eshan Henshaw and all other doctors in the Teaching Hospital, Calabar; Prof Emmanuel Daniel, Dr Ukott of the Teaching Hospital, Uyo for their assistance in the collection of blood samples from patients in the hypertension clinic. I sincerely thank Mr. Udoh (Fovic Medical Laboratory), Sis Theresa, Sis Affiong, Eneawan, Mr. Denise, Mr. Achibong, Mr. Etemma, Eka Ima, Eka Mmayeneka, Mrs. Essang and Mrs. Hansen who were relentless in taking me to their friends and relatives making it easier to collect samples from the general population. I also thank Mr. and Mrs. Kennedy-Kubiat for opening their door to me when I needed assistance. My brethren in Calabar, Ibadan and Abuja for their prayers. I really appreciate Dr Ben Igbinosa for helping me sort and analyze my data; Dr Jorge Franco for his assistance in analyzing my data; Dr kokoette Esenowo and Ms Iveren Chenge for their assistance. This page will not be complete without acknowledging my husband Esien Kooffreh, I thank God for giving him to me, his support both financially and otherwise has been immeasurable. My children Joseph, Emmanuel, David, Paul and Pearl kooffreh for their support and prayers. I know God will reward Ms Christiana Henry Asuquo for taking care of my home while I am away in Ibadan. I am deeply grateful to all and sundry who gave their blood samples and personal information to make this research a reality. iv Above all these, Thou Oh God who inhabits eternity, surely your favour and your tender mercies have indeed compassed me about. I bow my knees and say „Thank you‟ CERTIFICATION v I certify that this work was carried out by Mrs. M.E. Kooffreh in the Department of Zoology University of Ibadan and in the Virology Unit of the International Institute of Tropical Agriculture, Ibadan. …………………………………… Supervisor Chiaka.I. Anumudu B.Sc., (Benin) M.Sc. Ph.D. (Ibadan) Cellular Parasitology Programme, Dept of Zoology University of Ibadan, Nigeria. vi DEDICATION This work is dedicated to: The one who loved me and gave himself for me, My husband, Esien Ita Kooffreh, whose support has been overwhelming, My daughter Mary Esien Kooffreh who tried to grow up on her own and be a good girl, the years mummy was not there for her. And the boys, Joseph, Emmanuel, David and Paul Kooffreh vii TABLE OF CONTENTS Page Title page i Abstract ii Acknowledgements iv Certification vi Dedication vii Table of Contents viii List of Tables xii List of Figures xiii List of Plates xvi Abbreviations/Glossary/Definitions xvii CHAPTER ONE: 1.0 Introduction 1 1.1 Gene polymorphisms under consideration 4 1.2 Justification 7 1.3 Hypothesis 9 1.4 Objectives 9 CHAPTER TWO: 2.0 Literature Review 10 2.1 Pathophysiology of hypertension 10 2.2 Epidemiology of hypertension 12 viii 2.3 Association studies in hypertension 14 2.4 Mendelian forms of hypertension 17 2.5 Genes acting on pathways for BP regulation outside the RAAS 20 2.6 Mitochondrial genome mutation and essential hypertension 27 2.7 Genome-wide linkage analysis 29 CHAPTER THREE: 3.0 Materials and Methods 31 3.1 Study 31 3.2 Study Area 31 3.3 Subject and Enrolment 31 3.4 Collection of samples 32 3.5 Ethical approval 32 3.6 Questionnaire 36 3.7 Height measurement 36 3.8 Weight measurement 36 3.9 Blood pressure (BP) measurement 36 3.10 DNA extraction 36 3.11 Filter paper extraction of DNA 37 3.12 Preparation of agarose gel 38 3.13 Polymerase chain reaction – PCR 38 3.14 Enzymatic digestion 39 3.15 Protein A sandwich ELISA for the detection of plasma angiotensinogen 42 ix 3.16 Data management 43 CHAPTER FOUR 4.0 Results 44 4.1 Gel electrophoresis results 44 4.2 Demographic data 54 4.3 Genotypic frequencies 54 4.4 Blood pressure 55 4.5 Age 69 4.6 Knowledge of and family history of hypertension 69 4.7 Smoking and alcohol consumption status 69 4.8 Salt consumption 69 4.9 Educational attainment 79 4.10 Body mass index 79 4.11 Exercise 79 4.12 Marital status 79 4.13 Visits to fast food joints 79 4.14 Occupation 90 4.15 Regression analysis 90 4.16 Hardy-Weinberg Theory 90 4.17 Measurement of plasma angiotensinogen using sandwich ELISA 90 4.18 Analysis of Linkage disequilibrium 91 x CHAPTER FIVE: 5.0 Discussion 99 Conclusions 112 References 113 Appendices 136 xi LIST OF TABLES PAGE Table 2.1 Causative mutation for Mendelian forms of hypertension 19 Table 2.2 Summary of studies of candidate genes acting on pathways for BP regulation outside the RAAS 21 Table 4.1 Genotype and allele frequencies of the polymorphisms among the major ethnic groups in Calabar and Uyo 66 Table 4.2 Classification of the study population according to the JNC VII classification of blood 67 Table 4.3 Genotype and allele frequency of the RAAS and ANP polymorphisms in the patient and control population 68 Table 4.4 Characteristics of the individuals used in the protein A sandwich ELISA for the measurement of plasma angiotensinogen 96 Table 4.5 Linkage disequilibrium matrix for controls 97 Table 4.6 Linkage disequilibrium matrix for patients 98 xii LIST OF FIGURES PAGE Fig 1.1 The Renin Angiotensin Aldosterone and the Atrial Natriuretic peptide systems 8 Fig 2.1 Pathophysiologic mechanisms of hypertension 11 Fig 3.1 Map showing towns where samples were collected in South - south Nigeria 33 Fig3.2 Map showing sampling sites in Calabar city 34 Fig 3.3 Map showing sampling sites in Uyo city 35 Fig 3.4 The sequencing of the region which contains T174M and M235T Polymorphisms 40 Fig 4.1 Distribution by gender in the control and patient group 56 Fig 4.2 Distribution of the AGT M235T polymorphism by gender among the patient and control population 57 Fig 4.3 The ACE I/D allele distribution by gender in the control and patient population 58 Fig 4.4 The ATIR allele distribution by gender in the control and patient population 59 Fig 4.5 The ANP allele distribution by gender among the control and patient population 60 Fig 4.6 Ethnic distribution among the Control and Patient groups 61 Fig 4.7 Distribution of the AGT M235T polymorphism among the Ethnic groups in the Control population 62 Fig 4.8 Distribution of the AGT M235T polymorphism among the Ethnic groups in the Patient population 63 xiii Fig 4.9 Distribution of the ACE I/D polymorphism among the Ethnic groups in the Control population 64 Fig 4.10 Distribution of the ACE I/D polymorphism among the Ethnic groups in the Patient population. 65 Fig 4.11 Age distribution of patient and control group 70 Fig 4.12 Knowledge of hypertension status among patients 71 Fig 4.13 Smoking status among control and patient group 72 Fig 4.14 Alcohol consumption among control and patient groups 73 Fig 4.15 Distribution of the AGT M235T polymorphism and alcohol consumption in the study population. 74 Fig 4.16 Distribution of the ACE I/D polymorphism and alcohol consumption in the study population 75 Fig 4.17 Salt intake among control and patient groups 76 Fig 4.18 Distribution of the AGT M235T polymorphism and salt intake among the study population 77 Fig 4.19 Distribution of the ACE I/D polymorphism and salt intake among the study population 78 Fig 4.20 Educational levels among patient and control groups 80 Fig 4.21 Distribution of the AGT M235T polymorphism and Educational levels among the study population 81 Fig 4.22 Distribution of the ACE I/D polymorphism and educational levels among the study population 82 xiv Fig 4.23 Body Mass Index observed among control and patient groups 83 Fig 4.24 Exercise types observed among control group 84 Fig 4.25 Exercise types observed among patient group 85 Fig 4.26 Distribution of the ACE I/D polymorphism and exercise types among the study population 86 Fig 4.27 Distribution of the AGT M235T polymorphism and exercise types among the study population 87 Fig 4.28 Marital status of control and patient groups 88 Fig 4.29 Frequency of visits to fast food joints in the control and patient groups 89 Fig 4.30 Occupational groupings for patient population 92 Fig 4.31 Occupational grouping for control population 93 Fig 4.32 Distribution of the AGT M235T polymorphism and occupation among the study population 94 Fig 4.33 Distribution of the ACE I/D polymorphism and occupation among the study population 95 xv LIST OF PLATES PAGE Plate 4.1 Gel electrophoresis showing DNA quality 46 Plate 4.2 Gel electrophoresis showing 165 bp PCR product after amplification of the angiotensinogen gene 47 Plate 4.3 Agarose gel electrophoresis showing the amplification of the 165 bp fragment after enzymatic digestion with the Tth 111l restriction endonuclease enzyme 48 Plate 4.4 Agarose gel showing the amplification of the Insertion/Deletion of the angiotensin converting enzyme gene 49 Plate 4.5 Agarose gel electrophoresis showing the 850bp PCR product of the Angiotensin I type II receptor gene 50 Plate 4.6 Agarose gel electrophoresis showing digestion of the 850bp PCR product by the Dde1 restriction endonuclease 51 Plate 4.7 Agarose gel electrophoresis showing 157 bp PCR product of the atrial natriuretic peptide gene 52 Plate 4.8 Agarose gel electrophoresis showing the 157bp product after enzymatic natriuretic peptide gene 53 xvi ABBREVIATIONS AGT: Angiotensinogen EH: Essential hypertension RAAS: Renin-Angiotensin-Aldosterone System ANP: Atrial Natriuretic Peptide ACE: Angiotensin Converting Enzyme AT1R: Angiotensin II Type I receptor ELISA: Enzyme Linked Immunosorbent Assay BP: Blood Pressure BMI: Body Mass Index MM: Wild type allele for angiotensinogen with methionine at position 235 MT: Heterozygote allele for angiotensinogen with a threonine substitution at position 235 TT: Recessive homozygous allele for angiotensinogen with threonine replacing methionine at position 235 II: Insertion allele of the angiotensin converting enzyme gene ID: Insertion/Deletion heterozygote of the angiotensin converting enzyme gene DD: Deletion allele of the angiotensin converting enzyme gene AA: Wild type allele for the angiotensin II type I receptor gene with adenine at Position 1166 AC: heterozygous allele for the angiotensin II type I receptor gene with a cytosine Substitution at 1166 CC: Recessive homozygous allele for the angiotensin II type I receptor gene with a cytosine replacing adenine at position 1166 CC: Wild type allele for the atrial natriuretic peptide gene with cytosine at position 664 xvii CG: Heterozygous allele for the atrial natriuretic peptide gene with guanine substitution at position 664 GG: Recessive minor allele for the atrial natriuretic peptide gene with guanine Replacing cytosine at position 664 IITA: International Institute for Tropical Agriculture xviii CHAPTER ONE INTRODUCTION Hypertension is a multifactorial disorder that results from an interaction of many risk genes such as molecular variants of the angiotensinogen gene, angiotensin converting enzyme gene, angiotensin II receptor I gene and the corin gene (Cooper et al., 2000; Hilgers et al., 1999; Dries et al., 2005; Sethi et al., 2003), and environmental factors such as obesity, body mass index (BMI), dietary salt intake, alcohol consumption, stress and high-density lipid (HDL) – cholesterol levels. Genes determine approximately 20 to 60% of the variability in blood pressure in different populations (Sethi et al., 2003; Cooper et al., 2000). Hypertension is defined as high arterial blood pressure and it is generally agreed that blood pressure above 140/90 mm Hg is hypertension (Kadiri, 2000). Hypertension is a condition in which an increase in resistance to blood flow causes the blood to exert excessive pressure against the walls of the blood vessels. The heart must therefore work harder to pump blood through the narrowed blood vessels. If the condition persists, damage to the heart and blood vessels tend to increase the risk for stroke, heart attack, kidney or heart failure (Akinkugbe, 2000). Essential hypertension is high blood pressure for which no medical cause can be found and it accounts for more than 90% of cases of hypertension. The remaining 5-10% of hypertension also referred to secondary hypertension is due to other conditions that affect the kidneys, arteries, heart and the endocrine systems. Hypertension affects about one quarter of the adult population in industrialized countries and it contributes significantly to morbidity from stroke, heart failure, coronary heart disease and chronic kidney failure (Angius et al., 2002). Blood pressure is usually classified based on the systolic and diastolic blood pressure. Systolic pressure is the blood pressure in the vessels when the heart contracts or during heart beat. Diastolic pressure is the pressure in the vessels when the heart relaxes or between heart beats. Based on the recommendation of the seventh report of the Joint National Committee of Prevention, Detection, Evaluation and Treatment of High blood Pressure (JNC VII), the classification of blood pressure (expressed in mm Hg) for adults aged 18 or older is as follows: Normal - systolic lower than 120, diastolic lower than 80. Prehypertension - systolic 120-139, diastolic 80-90. Stage 1 hypertension – systolic 140-159, diastolic 90-99. Stage 2 hypertension - systolic equals or more than 160, diastolic equals or more than 100. The JNC VII report emphasizes that patients with prehypertension are at risk for progression to hypertension and that lifestyle modifications are important preventive strategies (Sharma and Kortas, 2008). These classifications are based on the average of an individual‟s blood pressure readings taken on two occasions. Diagnosis of hypertension is on the basis of chronic or persistent high blood pressure, it requires three separate sphygmomanometer measurements a week apart. A diagnosis for essential hypertension is made usually after all other forms of hypertension have been excluded using biochemical and clinical investigations. Mild to moderate essential hypertension is usually asymptomatic. Signs and symptoms associated with severe hypertension in adults and children include headache, drowsiness, fatigue, vision disorders, nosebleeds and facial paralysis, nausea and 1 vomiting. In infants and neonates, symptoms include failure to thrive, difficulty in breathing and seizures. (Rodriguez et al., 2010). Some other signs and symptoms suggest that hypertension is as a result of disorders in hormone regulation. These symptoms are specific to the disorder. An example is hypertension in pregnancy which is one of the symptoms of pre-eclampsia that usually progresses to eclampsia, a life threatening condition characterized by seizures. Although no direct cause has been identified as being responsible for essential hypertension, several factors have been shown to predispose individuals to high blood pressure. These include sedentary lifestyle, smoking, obesity, salt sensitivity, alcohol intake and vitamin D deficiency. The risk for essential hypertension also increases with age, some inherited genetic mutations, a family history of hypertension, an overactive sympathetic nervous system and increase in the levels of renin. Low birth weight has also been included as risk factor for adult essential hypertension. Secondary hypertension is usually due to an identifiable cause which if treated properly reduces the elevated blood pressure. Many other conditions also cause secondary hypertension, they include: hormonal imbalances, kidney disease, preeclampsia during pregnancy, certain prescriptions and illegal drugs such as decongestants and corticosteroids as well as some nutritional substances such as caffeine (McCrindle, 2010). Investigations into the molecular genetics of human hypertension is aimed at identifying the loci involved, detecting gene variants of these loci and associating them with intermediate phenotypes for proper estimation of their quantitative effect on blood pressure and interactions with principal environmental factors (Corvol and Jeunemaitre, 1997; Zhu et al., 2003; 2005). Evidence for a genetic influence on blood pressure comes from various sources. Twin studies have shown a greater concordance of blood pressures in monozygotic than dizygotic twins (Feinleib et al., 1977), and population studies show greater similarity in blood pressure within families than between families (Longini et al., 1984). The latter observation is not attributable to only a shared environment since adoption studies demonstrate greater concordance of blood pressure among biological siblings than adoptive siblings living in the same household (Biron et al., 1976). Furthermore, single genes can have major effects on blood pressure, accounting for the rare Mendelian forms of high and low blood pressure (Lifton et al., 2001). Although identifiable single-gene mutations account for only a small percentage of hypertension cases, the study of these rare disorders may clarify pathophysiologic mechanisms that predispose to more common forms of hypertension and may suggest novel therapeutic approaches (Lifton et al., 2001). Mutations in 10 genes that cause Mendelian forms of human hypertension and 9 genes that cause hypotension have been described to date, as reviewed by Lifton and colleagues (Lifton et al., 2001; Wilson et al., 2001). In most cases, hypertension results from a complex interaction of genetic, environmental, and demographic factors. Improved techniques of genetic analysis, especially genome-wide linkage analysis, have enabled a search for genes that contribute to the development of essential hypertension in the population. The application of these techniques has found statistically significant linkage of blood pressure to several chromosomal regions, including regions linked to familial combined hyperlipidemia (Hsueh et al., 2000; Kristjansson et al., 2002; Hunt et al., 2002). These findings suggest that there are many genetic loci, each with small effects on blood pressure in the general population. Overall, however, identifiable single-gene 2 causes of hypertension are uncommon, consistent with a multifactorial cause of essential hypertension (Oparil et al., 2003). The candidate gene approach typically compares the prevalence of hypertension or the level of blood pressure among individuals of contrasting genotypes at candidate loci in pathways known to be involved in blood pressure regulation. The most promising findings of such studies relate to genes of the renin– angiotensin–aldosterone system, such as the M235T variant in the angiotensinogen gene, which has been associated with increased circulating angiotensinogen levels and blood pressure in many distinct populations (Jeunemaitre et al., 1992b; Corvol et al., 1999; Staessen et al., 1999), and a common variant in the angiotensin-converting enzyme (ACE) gene that has been associated in some studies with blood pressure variation in men (Fornage et al., 1998; O’Donnel et al., 1998). However, these variants seem to only modestly affect blood pressure, and other candidate genes have not shown consistent and reproducible associations with blood pressure or hypertension in larger populations (Lifton et al., 2001); thus, demonstration of common genetic causes of hypertension in the general population remains elusive (Corvol et al., 1999; Niu et al., 1998; Luft, 2000). 1.1 Gene polymorphisms under consideration The AGT gene belongs to the serpin (serine protease inhibitor) super family (Corvol and Jeunemaitre 1997). The human AGT cDNA is 1,455 nucleotides long and codes for a 485-amino acid protein (Kageyama et al., 1984). The AGT gene contains five exons and four introns which span 13kb. In situ hybridization studies indicate that the human AGT gene is located on chromosome 1q42-43 (Isa et al., 1990; Gaillard –Sanchez et al., 1990). The human AGT protein is a globular glycoprotein with a molecular mass of 55-65 kDa, depending on the state of glycosylation. (Corvol and Jeunemaitre, 1997). A high molecular mass form of AGT protein is present in human plasma. In addition to the liver, the brain, large arteries, kidney and adipose tissues are all established sites of AGT synthesis (Dzau et al., 1987). The renin-angiotensin (R-A) system is a powerful pressure system which influences salt and water homeostasis. Angiotensinogen (AGT) is a key component of this system, it is cleaved by renin to yield angiotensinogen 1 (AGT 1), which is cleaved by angiotensinogen converting enzyme (ACE) to yield angiotensinogen II (AGT II), responsible for carrying out a range of functions that include i) prompting the constriction of blood vessels causing a rise in blood pressure, ii) ensuring the release of aldosterone by the adrenal cortex which acts on the tubules causing absorption of more water and salt from urine. Blood volume increases so does blood pressure. Potassium ions are excreted from the tubules in exchange for sodium iii) mediates the release of antidiuretic hormone from the pituitary that enhances the reabsorption of water, it also increases an individual‟s appetite for salt and stimulates the sensation of thirst (Caulfield et al., 1994). The M235T polymorphism was associated with a 10% - 30% increase in plasma AGT. Chronic increases in plasma AGT concentration may slightly increase blood pressure and facilitate hypertension (Corvol and Jeunemaitre, 1997). The measurement of angiotensinogen concentration has proved to be a convenient method for monitoring the activity of R-A system in human populations since it 3 circulates at relatively constant level. AGT is usually measured by an enzymatic assay for Ang 1 after its complete hydrolysis by excess renin. Direct immunoassays, using polyclonal and monoclonal antibodies against AGT, that measure both intact AGT and its inactive C-terminal part, residual AGT have also been developed (Genain et al., 1984; Katsurada et al., 2007). Cooper et al. (1999) that higher angiotensinogen levels were indicative of higher blood pressure. Simple and accurate methods to measure human angiotensinogen are available but very expensive. This study used one of the sandwich ELISA methods used by International Institute for Tropical Agricultural-IITA, Ibadan to detect plant viruses, the reagents are cheaper and if successful could be used to measure human angiotensinogen in plasma and also determine its concentration in the tropics to reduce cost. Angiotensin Converting Enzyme (ACE), a key enzyme in the renin angiotensin- aldosterone pathway, is found in the kidneys. It catalyzes the conversion of angiotensin I to a physiologically active angiotensin II that controls fluid electrolyte balance and systemic blood pressure (Wang et al., 2000). ACE gene is mapped to chromosome 17q23. The insertion/deletion (I/D) polymorphism was discovered in 1990 and is characterized by the presence of (insertion) or absence (deletion) of a 287- AluYa5 element inside intron 16 producing three genotypes: II homozygote, ID heterozygote, DD homozygote (Rigat et al., 1990). Though the polymorphism is located in a non- coding region of the ACE gene, several investigators (Sakuma et al., 2004; Salem, 2008; Tsezou et al., 2008; Ramachandran et al., 2008; Sameer et al., 2010) have observed that the polymorphism is not silent but the DD homozygote is associated with increased activity of ACE in the serum and several diseases including hypertension. The angiotensin II protein is a well known vasoconstrictor that exerts most of its influence through the angiotensin II type 1 receptor (AT1R). Angiotensin II type 1 receptor (AT1R) is a membrane-bound G protein coupled- receptor that mediates the effects of angiotensin II (De Gaspora et al., 2000). The highly polymorphic human AT1R gene is 55kb long having five exons and four introns, A1166C polymorphism is a single 1 base substitution of adenine for cytosine at position 1166 in the 3 untranslated regions of the gene located on chromosome 13. The A allele is the larger fragment that lacks the restriction enzyme while the smaller fragment from the C allele has the restriction enzyme site. The physiological significance of the polymorphism is uncertain because data on the function of the AT1R polymorphism is limited. Thus the mechanism responsible for the association of hypertension status with A1166C polymorphism has remained largely unknown and the amino acid sequence of the receptor is not altered. It is however thought to affect mRNA stability and transcription and is in linkage disequilibrium with some other polymorphism. It is also associated with some diseases (Bonnardeaux et al., 1994; van Geel et al., 2000; Stankovic et al., 2003; La pierre et al., 2006). In addition to the kidney, the heart plays an important role in regulating salt and water balance. This is mediated by a cardiac hormone referred to as the atrial natriuretic peptide (ANP) or factor (ANF). This is a potent natriuretic and vasorelaxant hormone that is mainly secreted by cardiomyocytes and plays a role in cardiovascular homeostasis in 4 opposition to the RAAS. When blood sodium and blood pressure levels increase, ANP secreted from the heart bind to its receptors in the kidney and blood vessels, promotes the excretion of large amounts of salt in urine, thereby lowering blood volume and also relaxing blood vessels. The heart and kidney are thus involved in maintaining a fine balance of electrolytes and body fluid. (Fig 1.1) The ANP is a 28 amino acid peptide in humans that assumes a hairpin structure by virtue of a cystein bridge that links residues 7 and 23. The ANP gene is located on chromosome 1q21. Several nucleotide polymorphisms have been identified in the ANP gene. One of them is the –C664G polymorphism located in the promoter region. Rubattu et al. (2006) reported that the – C664G polymorphism is responsible for the down regulation of ANP gene transcription; it is associated with left ventricular hypertrophy in Italians. The –C664G has been reported to be monomorphic among the Chinese and no other SNPs are in linkage disequilibrum with the –C664G polymorphism (Xue et al., 2008). 1.2 Justification Genetic variations of genes encoding components of the renin-angiotensin- aldosterone system have been associated with susceptibility to hypertension making them strong candidate genes for investigating the genetic basis of hypertension. In addition to the RAAS, the natriuretic peptide system also affects blood pressure directly through its vasodilatory and natriuretic activities and indirectly inhibiting the RAAS. This has generated considerable interest in the role of ANP in the development of hypertension. Some researchers have reported the frequency of the M235T and the T174M alleles of the angiotensinogen gene in western Nigeria. Most of the studies carried out so far in Nigeria have been concentrated in individuals and population in the South Western part of the country. Little is found in literature on the genetics of hypertension in other geographical and ethnic areas of Nigeria, and its genetic epidemiology, especially since variations in the dietary intake, culture (way of life) etc exist in these places. In Blacks, differences have been observed in the expression of risk factors such as level of circulating angiotensinogen, urbanization, dietary factors and gene variants for hypertension depending on the environment (Cooper et al., 1998; Cooper et al., 2005; Mufunda et al., 2006). 5 Fig 1.1 The Renin Angiotensin Aldosterone and the Atrial Natriuretic peptide systems 6 Apart from the M235T allele, there are no published data on other alleles in Nigeria. This study will provide baseline data for these areas and adds to the knowledge on the genetic basis of hypertension among these ethnic groups. The M235T polymorphism is associated with a 10-30% increase in plasma angiotensinogen. Chronic increases in plasma AGT concentration is thought to increase blood pressure and facilitate hypertension. Simple and accurate methods are available but very expensive. This study used one of the sandwich ELISA methods used by the International Institute for Tropical Agriculture Ibadan to test plant specimens for viruses, to measure the levels of angiotensinogen in human plasma and determine its concentration in relation to the presence of the T235 allele in the study population. 1.3 Hypothesis There is an association between the mutant variant of the genes for angiotensinogen; angiotensin converting enzyme; angiotensin II type I receptor; atrial natriuretic peptide (with possible interactions of environmental factors) and hypertension in the study population. 1.4 Objectives The main goal of this research was to improve our knowledge of the genetic epidemiology of hypertension in Calabar and Uyo. Specific objectives are: 1. To genotype the study population for: a) M235T allele of the angiotensinogen gene b) The A1166C allele of the angiotensin 11 type 1 receptor gene, c) The insertion deletion polymorphism of the angiotensin converting enzyme gene and d) The C664G allele of the atrial natriuretic peptide gene 2. To determine the association of these polymorphisms with hypertension status in a sample population of individuals living within the cities of Calabar and Uyo. 3. To relate the angiotensinogen levels in the plasma of hypertensives and controls to the M235T allele and to hypertension. CHAPTER TWO LITERATURE REVIEW 2.1 Pathophysiology of hypertension Hypertension results in a compromise of the pathophysiological mechanisms. The mechanisms associated with secondary hypertension are fully understood but those associated with essential hypertension are still unclear. However it is known that cardiac output is raised at the onset of the disease with total peripheral resistance (TPR) normal, as the disease progresses, cardiac output drops to normal with an increase in total peripheral resistance. Three theories have been put forward to explain this phenomenon: 1 Atrial natriuretic factor is secreted to promote the excretion of sodium by the kidney resulting in an increase in the total peripheral resistance. 7 2 Overproduction of sodium-retaining hormones and vasoconstrictors; long-term high sodium intake leading to vasoconstriction and retention of sodium and water; inadequate dietary intake of potassium and calcium. The result is an increase in blood volume that leads to hypertension. 3 Increased sympathetic nervous system activity, perhaps related to heightened exposure or response to psychosocial stress. Structural and functional abnormalities in the cardiac vasculature, including endothelial dysfunction, increased oxidative stress, vascular remodeling have been implicated in the development of hypertension, it is still unclear whether endothelial changes precede disease development or if such changes are due to chronic elevated blood pressure(Oparil et al., 2003) Figure 2.1. 8 Fig 2.1 Pathophysiologic mechanisms of hypertension (Oparil et al., 2003) 2.2 Epidemiology of hypertension High blood pressure or hypertension `a silent killer‟ condition is now the most common chronic condition affecting 20-30% of the adult population in the U.S. An estimated 63.3 million (31.0%) US adults currently have a BP exceeding 140/90mm Hg and the prevalence is higher for blacks than for other racial/ethnic subgroups. (Giles et al., 2007). National health surveys in various countries have reported a prevalence of 22% in Canada, of which 16% is controlled, 26.3% in Egypt of which 8% is controlled, 13.6% in China of which 3% is controlled (Sharma and Kortas, 2008). Ideal data on hypertension prevalence and incidence based on large population-based surveillance that use standardized and validated protocols are lacking for most countries in sub-Saharan 9 Africa. The aggregate of prevalence data published in the 1990s excluded highly selected groups in Africa with either very low or very high prevalence of 5-20%. In these studies, hypertension prevalence increased in a graded fashion with the established biological and psychosocial determinants of raised BP. Cooper et al., (1997) showed that hypertension was more common among the urban poor (17%) than among rural dwellers (7%). It was also substantially more prevalent among salaried suburban workers (26%). The effects of a family history of hypertension, alcohol intake, physical inactivity and advancing age in the prevalence of hypertension were similar to those established for developed nations although their relative importance as a cause of hypertension was different, though less evaluated in Africa. More recent data, such as those from Tanzania, Ghana, Nigeria, Egypt and South Africa suggest that hypertension prevalence (using a partition value of 140/90mm Hg) is on the rise in Africa and commonly exceeds 20 - 25% in rural areas and is over 30% in urban and semi urban areas (Addo et al., 2007). In a study of two linked cross sectional population- based surveys of a middle - income urban district and a relatively prosperous rural area in Tanzania, Edward et al. (2000) reported an age standardized hypertension prevalence of 37.3% and 39.1% among men and women respectively in the urban district, 26.3% and 27.4% among men and women respectively in the rural area. In a cross sectional study of adults age 18 years and older in four rural communities in the Ga district of Ghana, Addo et al. (2006) observed a hypertension prevalence of 25.4%. In particular, the age adjusted odds ratio for developing hypertension for overweight and for obesity were 5.8 and 6.9 respectively. Burkett (2006) reported a prevalence of 32.8% in a survey of volunteers in two villages in the Volta region of Ghana, more than 1 in 4 adults (28.7%) have high B.P with an even higher prevalence in semi urban areas (32.9%). Studies suggest that hypertension rises with age in both men and women, in adults as well as children and the prevalence tends to be higher in the urban than rural areas, though some populations in Africa still show relatively low hypertension prevalence (Kaufman et al., 1999; Cooper et al., 2005). Nigeria has a population of about 130 million people (National Census, 2006) and is the largest Black nation in the world. The crude prevalence of hypertension has been documented as 11.2% (based on blood pressure threshold of 160/95 mm Hg), with an age-adjusted ratio of 9.3%. This number translates into 4.33 million Nigerian hypertensives aged ≥15 years according to the National Census figures (Akinkugbe, 2000). However, according to the current definition of hypertension from the seventh Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure (JNC 7) guidelines (JNC 7, 2003) many more Nigerians (20–25%) would be classified as hypertensive. In a study carried out by the International Collaborative Study of Hypertension in Blacks (ICSHIB), the age-adjusted prevalence of hypertension in Nigeria was 14.5%. Based on gender, the prevalence of hypertension was 14.7% for men and 14.3% for women (Cooper et al., 1997). Ulasi et al. (2011) reported a prevalence of 42.2% among market traders at Ogbete in Enugu, Nigeria. Major target- organ complications of hypertension, such as left ventricular hypertrophy (Opadijo et al., 2003), diastolic dysfunction (Ike and Onwubere, 2003), congestive heart failure (Falase et al., 1983) ischemic heart disease (Falase et al., 1974) and renal failure (Akinkugbe, 1992) are well documented in Nigeria. Nigerians are particularly susceptible to 10 hypertension and its complications such as disabling and fatal strokes which remain a major cause of morbidity and mortality (Osuntokun et al., 1979; Iyalomhe et al., 2008). In a study of cardiovascular diseases in multiple centers in Nigeria (Ekere et al., 2005) hypertension was ranked first. Hypertension and its complications constitute <25% of emergency medical admissions in urban hospitals in the country (Iyalomhe et al., 2008). It is the medical illness most frequently diagnosed in elderly Nigerians (Bella et al., 1993). Ogunniyi et al. (2001) studied 613 elderly Nigerians (398 women and 215 men) aged 65–110 years in a cohort at Ibadan. They found that cardiovascular disease was the most common condition in this cohort, and hypertension (27.8%) was the most frequent diagnosis. Bella et al. (1993) also reported a similar figure. Ogah (2006) observed that hypertension was also the most common condition associated with dementia in Nigeria. It was the most common condition in senior executives (Okojie et al., 2000) and army recruits (Awoyemi et al., 2001). Two autopsy studies showed that hypertension is the most common cause of sudden unexpected natural death. Amakiri et al. (1997) studied 876 consecutive coroners‟ autopsies at Ibadan and found that the most common cause of sudden natural death was cardiovascular disease, and complications of hypertension accounted for most cases. This finding was corroborated by Aligbe et al. (1997) 3% of hypertensive Nigerians die each year. The population-attributable risk has been established at 7% (Kaufman et al., 1996). Low economic status was implicated in the development of hypertension among Nigerian adults (Adedoyin et al., 2005). 2.3 Association studies in hypertension RAAS (renin–angiotensin–aldosterone system) The human AGT gene (coding for angiotensinogen), an AGT precursor of the system, encodes a hepatic protein cleaved by renin and digested further by angiotensin- converting enzyme - ACE to generate the physiologically active angiotensin II (Ang II). Ang II via presynaptic type 1 Angiotensin II receptors - AT1Rs, potentiates the release of noradrenaline (norepinephrine). This peptide, together with aldosterone, which is generated in the adrenal zona glomerulosa by CYP11B2, maintains the circulating plasma volume that, in turn, through stimulation of cardiopulmonary and arterial mechanoreceptors, may influence sympathetic tone and increase heart rate variability (Grassi, 2001). Due to the important role of RAAS in the regulation of water and sodium balance (Naber and Siffert, 2004), numerous studies have investigated the relationship between RAAS and EH (Caulfield et al., 1995; Atwood et al., 1997; Brand et al., 1998; Salem, 2008; Badaruddoza et al., 2009; Sameer et al., 2010). Two molecular variants of the angiotensinogen gene, (M235T and T174M) were found to correlate with high levels of angiotensinogen in the plasma as well as hypertension (Sethi et al., 2003). These two variants are at some distance from both the cleavage sites for the promoter region, it is assumed that they are important in angiotensinogen transcription and messenger RNA stability (Corvol and Jeunemaitre, 1997). The AGT gene variant M235T is associated with higher circulating AGT levels and EH in several, but not all, populations (Jeunemaitre et al., 1992c; Atwood et al., 1997; Brand et al., 1998; Vasku et al., 2002). Support for linkage of M235T locus to essential hypertension was shown in studies on populations of European ancestry (Caulfield et al., 1994). Results from Japanese studies also show an association between 11 the angiotensinogen gene M235T variant and essential hypertension (Hata et al., 1994). Other studies have reported no association of the angiotensinogen variant with hypertension in African Americans and African Caribbeans (Caulfield et al., 1995; Rotimi et al., 1996; Sethi et al., 2001). The AGT M235T and the promoter G-6A polymorphisms showed association with essential hypertension in Tibetan women. No association could be detected for polymorphisms in ACE and AT1R with essential hypertension (Sun et al., 2004). The AGT M235T allele was demonstrated to be in linkage disequilibrium with allelic variants in the AGT promoter region (G-6A and A-20C), which may affect the basal rate of AGT transcription and could account for phenotypic variation in plasma AGT concentrations (Jeunemaitre et al., 1997; Inoue et al., 1997; Ishigami et al., 1997) . In Nigerians, Rotimi et al. (1997) reported a significant association between the presence of the M235T allele and high mean AGT concentration which was also significantly related to hypertension status, though Cooper et al. (1999) reported a high level of plasma angiotensinogen but low hypertension status in Igbo-Ora, Nigeria. The ACE gene was also implicated in the aetiology of hypertension. The gene- coding area carries an insertion/deletion - ID polymorphism within intron 16. Several studies have associated the ACE I/D polymorphism with elevation in blood pressure in the Japanese and other ethnic groups (Morise et al., 1994; Tobina et al., 2006 and Das et al., 2008) Some studies have shown that this polymorphism is strongly associated with increased blood pressure in males (O‟Donnell et al., 1998; Fornage et al., 1998; Sunder- Plassmann et al., 2002); however, a negative association was also detected in some linkage and association studies (Jeunemaitre et al., 1992a; Sugiyama et al., 1999). Gupta et al. (2009) reported a negative association between the ACE polymorphism and hypertension in a rural population in India. The relationship between essential hypertension and the genes encoding for AGT, ACE as well as AT1R was studied in 173 hypertensive individuals and 193 normotensive Chinese Tibetan individuals. The M235T and the G-6A polymorphisms showed association with essential hypertension in Tibetan women. However no associations were observed for polymorphism of the ACE and ATIR with essential hypertension. (Sun et al., 2004). The relationship between ACE and environmental factors predisposing to EH has been investigated in 1099 subjects from one Mongolian population. The study reported evidence for an interaction between the ACE DD (deletion/deletion) and ID polymorphism and cigarette smoking, alcohol drinking and BMI -body mass index (Xu et al., 2004). The ACE2 gene, a homologue of ACE, has been discovered (Donoghue et al., 2000), and appears to be a negative regulator of ACE in the heart (Eriksson et al., 2002). A case-control study investigating four single nucleotide polymorphisms - SNPs of ACE2 and EH provided no evidence for an association in an Anglo–Celtic Australian population (Benjafield et al., 2004). In this study, the 152 hypertensive subjects studied were the offspring of parents who both had hypertension, and similarly the 193 normotensive subjects were from normotensive parents over the age of 50 years. However, the data indicate little support for ACE2 in genetic pre-disposition to EH (Benjafield et al., 2004). As RAAS plays important roles in the regulation of water and sodium balance, the - adducin Gly460Trp variation is also believed to induce significant differences in the + + activity of the Na /K ATPase which, in the renal proximal tubule, affects sodium reabsorption (Lalouel et al, 2001). One study in a Chinese Han population (479 subjects 12 from 125 nuclear families) revealed that ACE ID, -adducin Gly460Trp and CYP11B2 - 344C/T polymorphisms interact to influence SBP (systolic BP; P<0.05), suggesting that these genes might indeed predispose to hypertension, especially in an ecogenetic context characterized by high salt intake (Wang et al., 2004). The A1166C polymorphism is associated with prevalent hypertension and increased aortic stiffness (Benetos et al., 1996; Wang et al., 1997; Danser and Schunket, 2000). This polymorphism has also been associated with other diseases such as left ventricular hypertrophy (Takami et al., 1998) pregnancy induced hypertension; early coronary disease and excessive vasoconstriction (Alvarez et al., 1998; van Geel et al., 1998; 2000). Stankovic et al. (2003) reported a significant association between this polymorphism and hypertension in males but not females. The frequency of the C1166 allele was high among hypertensives (Rubattu et al., 2004; Dzida et al., 2001). Some 1166 other studies have also reported a negative association between the A C polymorphism and hypertension (Tiret et al., 1998; Takami et al., 1998 and Kikuya et al., 2003). These variations were attributed to ethnic differences in the various populations (Agachan et al., 2003, Kikuya et al., 2003). Rubattu et al. (2007) found that young Italian men heterozygous for the G allele mutation of the ANP gene had an increased risk for an early onset of the disease. When compared with homozygous G individuals, carriers of the-664G mutation also had an increased left ventricular mass index in a study among a highly homogenous population of Caucasian patients (Rubattu et al., 2006). The C664G polymorphism showed a borderline association with hypertension in Japanese subjects (Kato et al., 2000). Hu et al. (2007) genotyped 1186 individuals from the Matsu area in Taiwan, 35 years and above, no difference was observed between the allele frequency of patients and controls. Zhang et al. (2006) also reported no association between the –C664G mutant and hypertension. Kato et al. (2002) did not observe any association between this polymorphism and stroke. 2.4 Mendelian forms of hypertension Molecular genetic studies have identified mutations in eight nuclear genes and one mitochondrial gene that cause Mendelian forms of hypertension (Table 2.1). They include CYP11B1/CYP11B2 -genes encoding steroid 11β hydroxylase/aldosterone synthase on chromosome 8p in glucocorticoid remediable aldosteronism - GRA (Lifton et al., 1992), + SCNN1B and SCNN1G are genes encoding for β and γ subunits of epithelial Na channel - ENaC respectively found on chromosome 16p in Liddle's syndrome (Shimkets et al., 1994; Hansson et al., 1995; Tamura et al., 1996; Melander et al., 1998), HSD11B2 gene encoding 11 β-hydroxysteroid dehydrogenase 2 - 11 β HSD2 on chromosome 16q in the syndrome of apparent mineralocorticoid excess - AME (Mune et al., 1995; Stewart et al., 1996), NR3C2 gene encoding mineralocorticoid receptor - MR on chromosome 4q in pregnancy induced hypertension (Geller et al., 2000). Also mutant genes of the serine/threonine protein kinases, WNK1 (lysine) protein kinase 1 on chromosome 12p and WNK4 on chromosome 17q, cause pseudohypoaldosteronism type II also known as familial hyperkalaemia and hypertension - FHH or Gordon's syndrome (Wilson et al., 2001). Extensive studies of FHH by Mayan et al. (2004) have found that affected subjects with WNK4 Q565E mutations have hypercalciuria accompanied by lower serum calcium 13 levels supporting a mechanism of renal calcium wasting. Together with the observation that WNK4 regulates the renal outer medullary potassium channel, as well as epithelial chloride/base exchange and the sodium/potassium/chloride co-transporter, an interaction between WNK4 and a calcium channel transporter was suggested (Mayan et al., 2004). Most of these disorders are due to defective genes acting in the same physiological pathway in the kidney, altering the net renal reabsorption of salt (Lifton et al., 2001). 14 Table 2.1 Causative mutation for Mendelian forms of hypertension Causative Enzyme Mode of Monogenic syndrome gene Characteristics of mutations function Inheritance Chromosome GRA CYP11B1 and Fusion gene arising from Increasing AD 8p CYP11B2 unequal crossover Truncation mutations in C- Pseudo-aldosteronism SCNN1B and terminal Increasing AD 16p (Liddle's syndrome) SCNN1G region and missense mutations FHH (Gordon's syndrome) WNK1 and Deletion and missense mutations Increasing AD 12p and 17q WNK4 A ME HSD11B2 Missense and deletion mutations Decreasing A R 1 6q Hypertension exacerbated NR3C2 Missense mutation Increasing AD 4q in pregnancy HTNB U nknown Unknown Unknown AD 12p Mit. Hypertension, MT-TI Missense mutation dysfunction Mit. Mit. hypercholesterolaemia and hypomagnesaemia Ile HSD11B2, gene encoding 11βHSD2; Mit., mitochondrial; MT-TI, mitochondrially encoded tRNA ; NR3C2, gene encoding MR; SCNN1B and SCNN1G, genes encoding β and γ subunits of epithelial sodium channel respectively. GRA – Glucocorticoid remediable aldosteronism, HTNB – Hypertension and brachydactyly, AME – Syndrome of apparent mineralocoticoid excess, FHH – Familial Hyperkalaemia and Hypertension (Gong and Hubner, 2006) 15 Hypertension and dyslipidaemia cluster more often than expected for the risk of many common cardiovascular diseases, i.e. myocardial infarction, congestive heart failure and stroke (Stergiou and Salgami, 2004). A cluster of metabolic defects caused by mutation in a mitochondrial tRNA was identified in one large Caucasian kindred by Wilson et al (2004). The kindred featured a cluster of hypertension, hypercholesterolaemia and hypomagnesaemia. Direct sequencing and single-strand conformational polymorphism (SSCP) analysis of the entire mitochondrial genome were performed. A novel mutation conferring a uridine cytidine transition was identified at I1e nucleotide 4291 of the mitochondrial tRNA - MT-TI gene. The mutation occurs I1e immediately 5´ to the tRNA anticodon. Uridine at this position is one of the most conserved bases. Biochemical studies with anticodon stem–loop analogues of tRNA have been performed and indicate that substitution of cytidine for uridine at this position markedly impairs ribosome binding (Ashraf et al., 1999). Thus Wilson et al. (2004) speculated that complexity can arise from a single mutation because of the combined effects of reduced penetrance and pleiotropy which underlines the value of studying very large kindreds. Another Mendelian form of hypertension, hypertension and brachydactyly 12 - HTNB has been mapped to a defined chromosomal region 12p (Schuster et al., 1996), but the molecular basis of the underlying defect is still not clear. This genetic region nearly overlaps with a later whole-genome-scan linkage analysis for essential hypertension in a large Chinese pedigree (Gong et al., 2003), which indicates the 12 susceptibility genes for essential hypertension may reside on chromosome 12p . Even though these rare syndromes with Mendelian inheritance only account for a small fraction of the pathological BP variation in the general population, they provide insight into the pathophysiology of hypertension. The identification of the molecular mechanisms of BP variation also has implications for the development and use of antihypertensive treatments that need not be restricted only to individuals with Mendelian forms of hypertension (Lifton et al., 2001). 2.5 Genes acting on pathways for BP regulation outside the RAAS An ever-expanding repertoire of genes outside of the RAAS has been tested for involvement in the genetic basis of essential hypertension (Table 2.2). A number of 16 Table 2.2 Summary of studies of Candidate genes acting on pathways for BP regulation outside the RAAS. Candidate Molecular basis in EH Polymorphisms Association with genes EH GRK4 Desensitization of G-protein-coupled receptors, including the R65L and A142V No D1 receptor in proximal tubules HSD3B1 The biosynthesis of steroid hormones, including aldosterone A486V Yes 338Leu T C No PTP1B Regulating insulin signalling via receptor dephosphorylation No Regulating insulin signalling via receptor dephosphorylation 1484insG No SLC9A3 Regulation of sodium reabsorption in the proximal tubule G1579A, G1709A, G1867A, C1945T, No A2041G and C2405T SCNN1B A key determinant of sodium homoeostasis ENaC G589S and ENaC i12-17CT Yes SCNN1G A key determinant of sodium homoeostasis ENaC V546l No GREB1 Depressor effect through the improvement of endothelial Yes in men dysfunction and modulation of sympathetic nerve activation -13945A T and 45718A G HPCAL1 Protection of neurons against calcium-induced death stimuli IMS-JST 126186 A C Yes in women in co-operation with neuronal apoptosis inhibitory protein BDKRB1 Activate the arachidonic acid nitric oxide cascade SNPs in NT 026437, 76646507, Yes in American 76623594 and 76647595 Caucasians BDKRB2 Activate the arachidonic acid nitric oxide cascade and affect -58C/T Yes in African the insulin-dependent glucose transport/utilization Americans SNP in NT 026437 76648043 Yes in American Caucasians CAT Reduce smooth muscle cell contraction and proliferation -844 A/G and -262 T/C Yes in Greek induced by endothelia, Ang II and a-adrenoreceptor agonists Caucasians AD, autosomal dominant; AR: autosomal recessive; HSD11B2, gene encoding 11β HSD2; Mit., mitochondrial; MT-TI, Ile mitochondrially encoded tRNA ; MOI, mode of inheritance; NR3C2, gene encoding MR; SCNN1B and SCNN1G, genes encoding β and γ subunits of ENaC respectively; EH, essential hypertension. (Gong and Hubner, 2006) 17 studies have shown a correlation between hyperinsulinaemia, insulin resistance and hypertension (Wang et al., 2004). Speirs et al. (2004) tested several novel potential candidates, namely, gene encoding G-protein-coupled-receptor kinase 4 - GRK4, gene encoding 3β-hydroxysteroid dehydrogenase/isomerase 1 - HSD3B1 and gene encoding protein phosphatase 1B - PTP1B genes in 168 Caucasian EH patients and 312 normotensive controls. The regulation of sodium excretion by the kidney is of paramount importance for homoeostasis of the extracellular fluid volume and thereby of arterial BP. GRK4 was implicated in human hypertension by desensitization of G-protein-coupled receptors, including the dopamine 1 - D1 receptor (Jose et al., 1998; Sanada et al., 1999; Jose et al., 2003). In humans with EH, there is a decrease in the responsiveness of the D1 receptor in proximal tubules due to the uncoupling of the D1 receptor from its G-protein–effector enzyme complex (Sanada et al., 1999; Jose et al., 2003). 3β - Hydroxysteroid dehydrogenase/isomerase 1 plays a role in the biosynthesis of steroid hormones, including aldosterone (Simard et al., 1996). It has been proposed that allelic variations in the HSD3B1 gene could lead to elevated plasma aldosterone, resulting in an increased intravascular volume and hypertension (Azizi et al., 1997). PTP1B negatively regulates insulin signaling via receptor dephosphorylation (Hashimoto and Goldstein, 1992). However, no association between variants in HSD3B1 and PTP1B genes and hypertension could be detected (Speirs et al., 2004). In contrast, the V allele of the A486V variant of GRK4 showed association with elevated BP (P=0.02 for EH). Zhu et al. (2004) studied the relationship between the SLC9A3 gene coding for sodium/hydrogen exchanger 3 - NHE and essential hypertension in 399 subjects of African or Afro–Caribbean origin (68% with essential hypertension) and 292 subjects Caucasian origin (50% with essential hypertension), trying to examine the relationship with hypertension and biochemical indices of sodium balance. Six variants were identified in total. NHE3 is a member of an increasing number of NHEs responsible for transport of sodium and hydrogen ions across the proximal tubule (Orlowski and Grinstein, 1997). Moreover, animal studies highlight that \this class of genes has potential importance in the control of BP (Schulthies et al., 1998a and 1998b; Aldred et al., 2000) however, no association between the variants was detected in EH patients from either African and Afro–Caribbean origin or Caucasian origin (Zhu et al., 2004). Gain-of-function mutations in the β- and γ-subunits of ENaC cause the monogenic form of hypertension known as Liddle's syndrome (Shimkets et al., 1994; Hansson et al., 1995; Tamura et al., 1996). A recent investigation in a Finnish population (Hannila- Handelberg et al, 2005) has shown a higher prevalence of three ENaC variants (βENaC G589S, βENaC i12-17CT and γENaC V546I) in 347 hypertensive patients compared with 175 normotensive individuals and 301 randomly chosen blood donors (P<0.01). When frequencies of the individual gene variants in the hypertensive patients were compared with those in the other two groups combined, only the frequency of the βENaC i12-17CT variant was significantly higher among the hypertensive patients than in the other two groups (P=0.001), whereas there was no significant difference in the prevalence of βENaC G589S and γENaC V546I variants 18 between the hypertensive and control groups. Patients carrying the three variant alleles also had an increased urinary potassium excretion rate in relation to their renin levels (P=0.034). However, no change in activity of the two ENaC amino acid variants was detected when they were expressed in Xenopus oocytes compared with wild-type ENaC (Hannila- Handelberg et al., 2005). Chromosome 2p24-p25 has been shown to be linked with hypertension in several studies (Zhu et al., 2001; Angius et al., 2002; Laivuori et al., 2003). A study was thus carried out in a Japanese general population investigating the association of polymorphisms in this region with BP. Forty-seven polymorphisms in 14 genes in the region between D2S2278 and D2S168 and in the region just outside of these two markers (between nucleotides 8845292–11946689) were genotyped in 1880 individuals, 796 of whom were hypertensive and 1084 normotensive (Kamide et al., 2005). Multivariate logistic regression analysis with adjustment for age, BMI, hyperlipidaemia, diabetes mellitus, smoking, drinking and antihypertensive medication identified 11 SNPs in three genes that were associated with hypertension using a dominant or recessive model (P<0.05). From them, only one SNP in the HPCAL1 gene coding for hippocalcin-like 1 in women (P=0.003) and two SNPs in the GREB1 gene coding for gene regulated by oestrogen in breast cancer 1 in men (P=0.008) had a significant association with susceptibility to hypertension and BP modulation (Kamide et al., 2005). SBP in women with the AA+AC genotype of the positively associated SNP IMS- JST126186 in the HPCAL1 gene was 16.7 mmHg higher that that with the CC genotype (P=0.003). HPCAL1 shares 94% amino acid identity with hippocalcin, 2+ which functions as a neuronal calcium sensor and possesses a Ca /myristoyl switch allowing it to translocate to the membrane (Mercer et al., 2000). The SBP in men with GG+GC genotypes of IMS-JST149391 in the GREB1 gene was 9.2 mmHg higher than in the men with the CC genotype (P=0.008), and was 9.2 mmHg higher in men with the AA+AG genotype of IMS-JST 149390 in the GREB1 gene than in those with the GG genotype (P=0.008). The two SNPs in the GREB1 gene were in tight linkage disequilibrium. GREB1 was identified as a direct target gene of oestrogen receptor - ERα and is evolutionarily conserved compared with mouse genome (Rae et al., 2005; Lin et al., 2004). Oestrogen has depression effects through the improvement of endothelial dysfunction (Yen et al., 2004) and modulation of sympathetic nerve activation (Brandin et al., 2004) in animal experiments. Oestrogen insufficiency may be related to postmenopausal hypertension (Dubey et al., 2002). Genetic variation in ER has been associated with coronary artery wall atherosclerosis and stroke (Shearman et al., 2003; 2005). ERs are required for normal vascular physiology in males (Mendelsohn et al., 2003) and oestrogen has direct vasodilator properties in men, as it does in women, but the relevance of this remains to be understood. Thus the authors concluded that GREB1 might play a role in BP regulation (Kamide et al., 2005). Genetic heterogeneity may exist in different populations for the genesis of hypertension. One association was assessed between the SNPs in the promoter region of the CAT gene (coding for catalase) and EH in Greek Caucasians and 19 African–Americans. An association was found with the specific genotype combination of CAT-844 homozygous AA together with CAT-262 CT or TT in Caucasians only (100 hypertensive and 93 normotensive subjects; P=0.0339), whereas no association was observed in African–Americans -129 hypertensive and 98 normotensive subjects (Zhou et al., 2005). The role of oxidative stress in hypertension has been tested in a number of studies (Trouyz and Schiffin, 2004). Catalase, an enzyme H2O2 into water and oxygen, has been shown to reduce smooth muscle cell contraction and proliferation induced by endothelia, Ang II and -adrenoreceptor agonists (Wassman et al., 2004). Experimental studies have shown a protective role of higher catalase expression levels in hypertensive animal models (Uddin et al., 2003; Yang et al., 2003). Similarly, one SNP in the BDKRB2 gene coding for bradykinin receptor B2 and three SNPs in the BDKRB1 gene coding for bradykinin receptor B1 were associated with hypertension in American Caucasians (n=220; P values were between 0.026 and 0.0004). One SNP in the promoter region of BDKRB2 gene was associated with hypertension in African–Americans [n=218; P=0.044] (Cui et al., 2005). Bradykinin has a variety of vasoactive and metabolic effects, including vasodilatation via interaction with components of the arachidonic acid cascade (Merkus et al., 2002). Bradykinin enhances insulin-independent glucose transport through B1 and B2 receptors. Genetic variations of these receptors may alter the functional capacity of bradykinin, which may in turn alter an individual susceptibility to hypertension. The results described above suggest that individual SNPs may not be as important as the interaction among several SNPs. The genetic factors that contribute to hypertension are likely to be different among different ethnic populations. Further studies of association in a large number of genes in different pathways will be required to identify the possible interaction among genes and the full array of genetic factors causing hypertension. Epidemiological studies show that the risk of cardiovascular disease mortality and morbidity is much higher in hypertensive patients compared with normotensive people. Whether the genetic variants causing hypertension have an additive effect on pathways related to the risk for developing end-organ damage has been studied by Fabris et al. (2005) who examined variants from AGT M235T, ACE ID, AT1R A1166C and CYP11B2 -344C/T in 86 hypertension patients with renal insufficiency and 172 hypertensive patients without renal damage matched for age and hypertension. The cohort was followed for 2 years and investigated whether these variants may act synergistically to confer an increased risk for renal failure in hypertensive patients. AGT TT/AT1R AC (P=0.0018) and CYP11B2 CC/ACE DD (P=0.0012) showed a positive interaction in the development of renal insufficiency among hypertensive patients, and the association of AGT MM/AT1R AA (P=0.04) and AGT MM/AT1R AA/CYP11B2 TT (P=0.04) or AGT MM/AT1R AA/CYP11B2 TC (P=0.03) combinations were associated with a reduced risk of renal failure. Fabris et al. (2005) then concluded that, in patients with essential hypertension, an unfavourable allelic pattern of components of the RAAS may contribute to the increased risk for the development of renal failure. 20 Lipocalin-type prostaglandin D synthase - L-PGDS is a secretory protein of the lipocalin superfamily, which synthesizes prostaglandin D2 - PGD2 from prostaglandin H2 - PGH2 (Urade et al., 1985). Patients with hypertension exhibited a higher level of L-PGDS in serum and urine (Hirawa et al., 2002). A group of Japanese researchers investigated the association between its variants and the severity of carotid arteriosclerosis in 782 EH patients. The study by Hirawa et al. (2002) suggested that the 4111A C polymorphism in the PTGDS gene (coding for L-PGDS) is associated with the severity of carotid arteriosclerosis (P=0.002) and inversely correlated with increased high-density lipoprotein - HDL cholesterol (P<0.001) in Japanese hypertensive patients. However, the other variants had no relationship with the phenotype studied. The functional mechanism of the 4111A/C in L-PGDS remains unknown (Miwa et al., 2004). A small cross-sectional study in 140 normotensive subjects was carried out to ascertain the relationship between the polymorphism of the GSTM1 gene coding for glutathione S-transferase – GST M1, BP level and exposure to cigarette smoking. For analysis, the combination of genotypes, sex and smoking behaviour were used as qualitative variables, and age, BMI and heart rate were used as covariates. The combination „present-GSTT1 (GST theta 1), null- GSTM1‟ genotypes odds ratio - OR, 0.001; 95% CI, 0.00–0.439; P=0.025], heart rate (OR, 1.065; 95% CI, 1.018–1.114; P=0.006) and interaction between BMI and combination of „present-GSTT1, null-GSTM1‟ genotypes (OR, 1.319; 95% CI, 1.058–1.644; P=0.014) was detected to be associated with SBP. The results suggested that the GSTM1 gene is one of the candidate genes altering baseline SBP in normotensive individuals when the age, sex and smoking behaviour were considered (Saadat and Dadbine- Pour, 2005). The GSTs are involved in the detoxification of many toxic compounds of different chemical structures in cigarette smoke (Ketterer et al., 1992), whereas cigarette smoking is one of the major risk factors to cardiovascular diseases (Kannel et al., 1990). 2.6 Mitochondrial genome mutations and Essential Hypertension DeStefano et al. (2001) studied maternal and paternal effects in the development of human essential hypertension in American Caucasians, Greek Caucasians and African–Americans. They observed that, among parents with known hypertensive status, the proportion of affected mothers was significantly higher than the proportion of affected fathers in all three ethnic groups (DeStefano et al., 2001). The fraction of patients with EH potentially due to mtDNA (mitochondrial DNA) mutation involvement was estimated at 55% [95% CI, 45– 65%] (Sun et al., 2003). A complete sequencing of the mitochondrial genome from 20 hypertensive probands in African–American (n=10) and Caucasian families (n=10) was carried out. A total of 297 bp exchanges were identified, including 24 in rRNA genes, 15 in tRNA genes and 46 amino acid substitutions, with the remainder involving the non-coding regions (Schwartz et al., 2004). Several of these have been associated with cardiovascular and renal pathologies in previous studies (Watson et al., 2001; Khogali et al., 2001). Among them, an A10398G mutation in the MT-ND3 gene (coding for mitochondrially encoded 21 NADH dehydrogenase 3), identified in 12 hypertensive individuals of both ethnic groups, had been shown to occur with increased frequency in African–Americans with EH associated with end-stage renal disease (Watson et al., 2001). UCPs (uncoupling proteins) are inner mitochondrial-membrane-associated proteins and act as proton channels or transporters. Although mitochondria uses energy derived from fuel combustion to create a proton electrochemical gradient across the mitochondrial inner membrane, UCPs uncouple proton entry in the mitochondrial matrix from ATP synthesis (Boss et al., 2000). The functional polymorphism (-866 G/A) in the UCP2 promoter has been reported to be associated with obesity in an analysis of 340 obese and 256 never- obese middle-aged Caucasian subjects (P=0.007) (Esterbauer et al., 2001). Another association study between this polymorphism, obesity, and hypertension as well as Type II diabetes mellitus was carried out in a Japanese population with 342 Type II diabetic patients (among them 158 patients complicated with hypertension), 156 hypertensive patients without diabetes mellitus and 134 control subjects. The polymorphism was detected to be significantly associated with hypertension (frequency of A allele, 51.8% in hypertensives compared with 46.6% in normotensives; P<0.05), but was not associated with obesity in the Japanese population, which is in contrast with the significant association with obesity in Caucasians (Ji et al., 2004). Mitochondrial coupling factor 6, an essential component of mitochondrial ATP synthase, suppresses the synthesis of prostacyclin in vascular endothelial cells (Knowles et al., 1971). The role of the gene was studied in spontaneous hypertensive rats - SHRs (Osanai et al., 2001). In vivo, the peptide circulates in the rat vascular system, and its gene expression and plasma concentration are higher in SHRs than in normotensive controls. Functional analysis suggests it acts as a potent endogenous vasoconstrictor in the fashion of a circulating hormone c). Circulating coupling factor 6 is elevated in human hypertensive patients (n=30) compared with normotensive subjects (n=27; P<0.01) and was increased after salt loading in hypertensive patients. The percentage changes in plasma coupling factor 6 level after salt restriction and loading were positively correlated with those in mean BP (r=0.57; P<0.01) and negatively correlated with those in plasma nitric oxide level [r=-0.51; P<0.05] (Osanai et al., 2001; 2003 ). The elevated circulating coupling factor 6 in SHRs and human hypertension patients indicates that it is involved in the regulation of arterial BP in physiological and pathological conditions (Osanai et al., 2001; 2003). All of the above studies suggest that essential hypertension may not be only polygenic, but may also a „polygenomic‟ disorder. 2.7 Genome-wide linkage analysis Genome-wide linkage analysis predicts that multiple chromosomal regions may play a role in the development of human EH; however, lack of consistency across studies makes it difficult to draw any general conclusion for the genetic cause of human EH (Gong et al., 2003; Angius et al., 2002; Harrap et al., 2003). An investigation focusing on only SBP and DBP (diastolic BP) in 1109 white female dizygotic twin pairs has been carried out (de Lange et al., 2004). No 22 significant linkage with BP could be detected in this study, but several suggestive linkage regions were replicated and one novel suggestive linkage region for SBP on chromosome 11p was detected (de Lange et al., 2004). Significant linkage for longitudinal SBP from the Framingham Heart study was detected on chromosome 1q. In this study (James et al., 2003), the SBP for each individual was modelled as a function of age using a mixed modelling methodology; it was thus the best linear unbiased predictor of the individual's deviation from the population rate of change in SBP for each year of age whilst controlling for gender, BMI and hypertension treatment. Two previous linkage studies of hypertension had found peak LOD (logarithm of the odds) scores in the same region ( Hunt et al., 2002). Similarly, linkage on chromosomes 12q, 15q and 17q for mean SBP and linkage for both SBP slope and curvature on chromosome 20q were detected in the other study of the data from the Framingham Heart study (Jacobs et al., 2003). The linkage analysis for age at diagnosis of hypertension and early-onset hypertension in the HyperGEN (Hypertension Genetic Epidemiology Network) cohort of different populations was carried out (Wilk et al., 2004). Several suggestive linkage loci were detected and some of them have been reported to be linked to hypertension and BP in previous studies. These encouraging results suggest that linkage can be replicated from other studies and, therefore, new genetic factors with moderate-to-large effects can potentially be discovered. Considering the power of the individual studies, two genome-scan meta-analysis for hypertension were carried out individually (Liu et al., 2004; Koivukoski et al., 2004). Interestingly, the previous meta-analysis with different populations (Kristjansson et al., 2002; Perola et al., 2000; Ranade et al., 2003) failed to detect significant linkage to hypertension, only several regions with suggestive linkage were identified, including chromosomes 2p, 5q, 6q, 8p, 9p, 9q and 11q. From them, only regions on chromosomes 5q, 6q and 11q had P<0.05 (Liu et al., 2004). Controversially, meta-analysis of genome-wide scans for hypertension and blood pressure in Caucasians (Hunt et al., 2002; Kardia et al., 2003; Von Wowern et al., 2003) had significant linkage on chromosomes 2p12-q22 and 3p14-q12( Koivukoski et al., 2004). The results strongly suggest a population difference in the common phenotype. The mixed populations probably have a considerable degree of genetic heterogeneity, which is one of the main reasons why pooling of the results in different populations in the meta-analysis did not enhance the signals. However, pooling of the results in Caucasians possesses a smaller degree of genetic heterogeneity. One admixture mapping for hypertension loci with genome-scan markers was carried out in African–Americans (Zhu et al., 2005) using individuals from Nigeria as African ancestral population and European–Americans for the estimates of allele frequencies for European ancestors. The distribution of marker- location-specific for African ancestry was shifted upwards in hypertensive cases compared with normotensive controls, and the markers were located on chromosome 6q24 and 21q21. 23 24 CHAPTER THREE MATERIALS AND METHODS 3.1 Study A population based association – case control – study was used to determine the frequency of M235T allele, ACE I/D allele, AT1R allele, ANP gene and its relationship with the hypertension status in two populations in Akwa Ibom and Cross River States. The study also measured angiotensinogen levels in the plasma using the enzyme linked immunosorbent assay to relate it to the M235T allele and hypertension. 3.2 Study Area Cross River and Akwa Ibom states are sister states, both are part of the old Calabar kingdom Fig 3.1. Cross River State is a coastal state in South-South Nigeria, named after Cross River which passes through the state. It is located in the Niger Delta. Calabar is the state capital Fig 3.2. The state occupies 20,156sq kilometers. It shares boundaries with Benue state to the north, Enugu and Abia states to the west, to the east by Cameroon republic and to the south by Akwa Ibom and the Atlantic Ocean. The three major languages are Efik, Ejagham, and Bekwara. These different groups though distinct bear striking similarities in their cultures. Official population figures stand at 2,888,966 (National Population Census, 2006). Akwa Ibom State is also located in the coastal South-South of the country, lying o o o o between latitudes 4 321 and 5 331 north, and longitudes 7 251 and 8 251 east. Uyo is the state capital Fig 3.3. The state is bordered on the east by Cross River state, on the west by Rivers and Abia states, on the south by the Atlantic Ocean. Akwa Ibom has a population of 3,920,208 million according to the 2006 population census estimates. Along with English, the main languages here are Ibibio, Annang, Oron, Ibeno and Eket. 3.3 Subject and Enrolment A total sample population of 1,224 adult men (497) and women (727) from different ethnic groups were included in this study. The participants were grouped based on their ethnicity, but not on the basis of location. Of this number, 612 were patients attending the hypertension clinics in the University of Calabar Teaching Hospital, Calabar, the University of Uyo Teaching Hospital, Uyo and the General Hospital, Calabar. The other 612 were individuals whose blood pressure was below 140/90mmHg, who were not taking hypertensive drugs and not below the age of twenty from the same population. These individuals served as the control group. Inclusion criteria: All patients were individuals whose BP were consistently above 140/90 mmHg or were taking hypertensive medications. Controls were individuals whose BP were consistently below 140/90 mmHg and were not taking hypertensive medications. Exclusion criteria: females in the population using oral contraceptives were excluded from the study population. 3.4 Collection of samples Venous blood (3ml) was collected from each participant after they had given informed consent, into bottles containing anticoagulant EDTA Some participants refused blood collection from the upper arm; blood was obtained from thumb pricks and blotted 25 onto a chromatography (Whatman, no 3) paper, allowed to dry at room temperature and preserved in plastic bags prior to DNA extraction.. Plasma was obtained by centrifugation of blood samples. Blood and plasma was kept frozen in the freezer/cold room and transported by air to the Department of Zoology University of Ibadan (in coolers with ice o packs) where samples were kept at -70 . Samples were then transferred on wet ice to freezers in the International Institute of Tropical Agriculture, Ibadan for subsequent lab investigations. DNA was extracted from blood for genotyping of the polymorphism. 3.5 Ethical Approval Subjects included in the study gave informed consent and ethical approval for the study was obtained from the joint UI/UCH ethical review committee and each of the health establishment concerned- University Teaching Hospital Calabar, University of Uyo Teaching Hospital and the General Hospital, Calabar. 26 Fig 3.1 Map showing towns where samples were collected in south- south Nigeria 27 Fig 3.2 Map showing sampling sites in Calabar city 28 Fig 3.3 Map showing sampling sites in Uyo city. 29 3.6 Questionnaire Questionnaires were developed in English to assist in acquiring information on family history for hypertension and socio-demographic data, age, sex, dietary habits, physical activity, smoking habits and alcohol consumption (which could predispose an individual to hypertension). Informed consent was translated into Efik and Ibibio dialects to assist in explaining the research to the participants. 3.7 Height Measurement The wall in the collection centre was calibrated in meters. Individuals stood without foot or head wear facing the investigator, looking straight ahead and the investigator placed a ruler on top of the head of the individual and the reading in meters was recorded. 3.8 Weight Measurement Using the conventional weight scale, weight was measured in kilograms. 3.9 Blood Pressure (BP) Measurement Readings was taken using a sphygmomanometer in millimeters of mercury by certified medical personnel for the patients in the clinics and a certified nurse for the controls in the general population. Systolic and diastolic BP values were recorded. Before taking the measurement, the respondent was advised to sit quietly for 5 mins, with the legs uncrossed and the right hand free from clothing. The right hand was placed on the table with the palm facing upwards. The appropriate cuff size was selected and the cuff wrapped and fastened securely. The cuff was kept at the same level as the heart during measurement. The upper reading, the systolic blood pressure (SBP) and lower reading the diastolic blood pressure (DBP) recorded, the first and second reading were taken twice and the average of the two used for the analysis. 3.10 DNA Extraction DNA was extracted according to Dellaporta, (1983) with some modifications. Packed red blood cell after plasma had been removed (150μl) was put into 1.5ml eppendorf tube placed on a rack. Extraction buffer (350μl) was then added to the tube. Sodium dodecyl sulphate -SDS (40μl of the 20% solution) was added and the tubes were o inverted 3-4 times before they were incubated in a water bath at 65 C for 10 mins. The tubes were brought out of the incubator and left on the bench to cool to room temperature. Potassium acetate (160μl of the 5M solution) was added to the tubes. The tubes were inverted 2-3 times and centrifuged at 10,000rcf for 10 mins. About 400μl of the supernatant was carefully transferred into new eppendorf tubes and 200μl of cold isopropanol was then added. The tubes were inverted gently 5-6 times to precipitate o DNA. The tubes were kept on ice or stored in the fridge at 4 C for 15-20 mins. The tubes were then centrifuged at 10,000 rcf for 10 mins to sediment the DNA. The supernatant was decanted gently in order to ensure the pellet was not disturbed, 500µl of cold ethanol was added to the pellet to wash the DNA, then centrifuged at 10,000rcf for 5-10 mins. The ethanol was decanted and the DNA was air-dried at room temperature until no trace of alcohol was seen in the tubes. 30 Modifications to the original plant DNA extraction method originally developed by Dellaporta, (1983) are: 500µl of extraction buffer was added to 50mg of ground leaves of a plant; Plant samples are usually vortexed before incubation in the water bath and after the addition of 5M potassium acetate and 33µl of sodium dodecyl sulphate was added to the mixture of the ground leaf and extraction buffer. The DNA was re – suspended in 50μl of Tris-EDTA – T.E buffer and stored in the freezer as stock solution. An aliquot of the DNA stock solution was run on agarose gel electrophoresis to check the quantity of DNA. Each sample of genomic DNA (5μl) was mixed with 2ul of loading dye and transferred into the wells of the gel. A voltage of 110V was applied for about 45 mins. The samples were scored faint, +, 2+, 3+ depending on the intensity of the bands to indicate the amount of DNA extracted. Plate 3.2 3.11 Filter paper extraction of DNA This was done according to Bereczky et al. (2005). Pieces (1-2) of the filter paper about 5mm in diameter were cut using a sterile blade for each samples. These pieces were placed in an eppendorf tube, soaked in 65µl of T.E buffer. The tube was incubated at o 50 C for 15 mins in a water bath. The pieces were pressed gently at the bottom of the tube several times using a new pipette tip for each sample. o The eppendorf tubes were heated again for 15mins at 97 C to elute the DNA. The liquid condensing on the lid and the walls of the tube were removed by centrifuging for o 2-3 secs.The DNA extract that is the supernatant was kept at -20 C before use. 3.12 Preparation of agarose gel The ends of the plastic tray supplied with the electrophoresis apparatus was sealed with adhesive tape. 100ml of 1 TAE was measured and put into a bottle; 1.5g of agarose powder was added and heated in the microwave oven for 5 min to dissolve the agarose. o The bottle was then allowed to cool to about 60 C; 5µl of ethidium bromide was added. When checking for DNA quality, 0.8g of agarose and 0.8ul of ethidium bromide was used. The agarose was poured into the tray and allowed to set at room temperature (30 – 45 mins). The comb and tape were removed and the gel was mounted in the electrophoresis tank.1 TAE buffer was added enough to cover the gel. The samples of DNA were mixed with loading dye and slowly loaded into the wells of the submerged gel. For PCR, the buffer already contained the dye. The amplicons were loaded straight into the wells. A voltage of 110V was applied for 30 – 45 mins until the bromophenol blue had migrated an appropriate distance from the gel. The current was turned off and the gel was examined under ultraviolet light. 3.13 Polymarase Chain Reaction - PCR: Stock DNA samples were diluted 1/10, 1/50, 1/100 depending on the amount of DNA in the sample checked after extraction. A 1/10 dilution was done for all filter paper extracted DNA stock solution. Samples that were not amplified in the gel at the first PCR were repeated. M235T allele of the angiotensinogen gene Genomic DNA (2μl) was amplified in a 15µl reaction mix containing Go Taq® green master mix (Promega) 7.5µl, upstream and downstream oligonucleotide primers 0.30µl each and 4.9μl of nuclease-free water. 31 Cycling conditions: Below is the primer sequence used for these experiments, an o o initial denaturation for 10 mins at 95 C was followed by 35 cycles of 1 mins at 94 C, 1 o o o mins at 56 C and 1 min 30 secs at 72 C and a final elongation of 10 mins at 72 C. Primer sequence (Procopciuc et al, 2002) ' ' 5 CAGGGTGCTGTCCACACTGGACCCC 3 ' ' 3 CCGTTTGTGCAGGGCCTGGCTCTCT 5 3.14 Enzymatic digestion The specific mismatch incorporated into the antisense primer creates a Tth 111l site if the T235 variant is present fig 2.2. The presence of a cytosine at position 704; GACN↓NNGTC, the enzyme cuts to give a 141 and a 24 bp fragment. For the 235M variant, presence of thymine at position 704; GATNNNGTC, the enzyme does not cut the fragment leaving a 165 bp fragment with no restriction fragments (Basak et al, 2008). A cocktail of 0.25ul of the Tth111I enzyme, 1.5ul of the 10 x buffers and 3ul of sterile water was added to 10ul of the PCR product. The enzyme digestion was performed in a o final volume of 14.75ul at 65 C for 2 hours. The digested products were separated on 3% agarose gel stained with 1.2ul of ethidium bromide for 30 mins at 125V. 32 ENZYMATIC DIGESTION M235T 25-Mer MBI sense: (5‟-CCG TT GTG CAG GGC CTG GCT CTC T-3‟) and 25-Mer MBI antisense: (5‟-CAG GGT GCT GTC CAC ACT GGA CCC C-3‟). T GGC ACC CTG GCC TCT CTC TAT CTG GGA GCC TTG GAC CAC ACA GCT GAC AGG CTA CAG GCA ATC CTG GGT GTT CCT TGG AAG GAC AAG AAC TGC ACC TC CGG CTG GAT GCG CAC AAG GTC CTG TCT GCC CTG CAG GCT GTA CAG GGC CTG CTA GTG GCC CAG GGC AGG GCT GAT AGC CAG GCC CAG CTG CTG CTG TCC↓ AC(/T)G GTG GTG GGC GTG TTC ACA GCC CCA GGC CTG CAC CTG AAG CAG CCG TT GTG CAG GGC CTG GCT CTC TAT ACC CCT GTG GTC CTC CCA CGC TCT CTG GAC TTC ACA GAA CTG GAT GTT GCT GCT GAG AAG ATT GAC AGG TTC ATG CAG GCT GTG ACA GGA TGG AAG ACT GGC TGC TC CTG ATG↓GG(AG)→(GT)CC AGT GTG GAC AGC ACC CTG Fig 3.2 The sequencing of the region which contains T174M and M235T polymorphisms. (Basak et al., 2008). The italic letters mark the sequence corresponding to the primers used for T174M, the underlined letters- for M235T primers, and the bold letters represent the restrictions sites. This PCR cocktail was used for the ACE I/D allele. Genomic DNA (2μl) was amplified in a 12.5µl reaction mix containing Promega flexi green buffer 2.5µl, dNTPs 0.25µl, upstream and downstream oligonucleotide primers 0.25µl each, magnesium chloride 0.75µl, 6.44μl of nuclease-free water and Taq DNA polymerase 0.06µl. o Cycling conditions: An initial denaturation for 5 mins at 94 C was followed by o o o 30 cycles of 45 secs at 94 C, 45 secs at 56 C and 45 secs at 72 C and a final o elongation of 10 mins at 72 C. (I/D) POLYMORPHISM OF THE ACE GENE: Primer sequence Sense 5'- CTG GAG AGC CAC TCC CAT CCT TTC T -3' Antisense 3'- GAC GTC GCC ATC ACA TTC GTC AGA T -5' For the angiotensin 11 type 1 receptor gene and atrial natruiretic peptide gene that require RFLP digestion to identify alleles, Genomic DNA (4μl) was amplified in a 25µl PCR reaction mix containing Promega flexi green buffer 5µl, dNTPs 0.5µl, upstream and downstream oligonucleotide primers 0.5µl each, magnesium chloride 1.5µl, 12.88μl of nuclease-free water and Taq DNA polymerase 0.06µl. 33 o AT1R gene: Cycling conditions include an initial denaturation at 94 C for 2 mins, o o followed by 40 cycles of a further denaturation at 94 C for 1 min, annealing at 60 C o o for 1 min, extension 72 C for 2mins, and a final extension of 72 C for 10 mins. A1166C Polymorphism of the Angiotensin 11 Type 1 Receptor primer sequence 5' - AAT GCT TGT AGC CAA AGT CAC CT- 3' 5' - GGC TTT GCT TTG TCT TGT TG -3' A cocktail of 0.25µl of the Dde1 enzyme, 1µl of the 10 x buffer D; 0.1µl of acetyl BSA and 8.5µl of sterile water was added to 10µl of the PCR product. The o enzyme digestion was performed in a final volume of 19.85µl at 37 C for 4 hours. For the Rsa1 enzyme, the same concentration was used in preparing the cocktail but Rsa1 enzyme and 10x buffer E were substituted. The digested products were separated on 2% agarose gel stained with 10µl of ethidium bromide for 30 mins at 125V. o ANP gene: Cycling conditions include an initial denaturation at 95 C for 3 o mins, followed by 35 cycles of a further denaturation at 94 C for 20 secs, annealing at o o o 60 C for 30 secs, extension 72 C for 30 secs, and a final extension at 72 C for 5 mins. 664 C G Polymorphism of the Atrial Natriuretic Peptide Gene primer sequence 5' – AAC AGC AAC GGA AGA AAT GA -3' 5' – ATC CAA CCC CCA AAT AGA AGT A-3' 3.15 Protein A sandwich Enzyme Linked Immunosorbent Assay (ELISA) for the detection of plasma angiotensinogen. Principle: The protein A sandwich ELISA procedure uses protein A to increase the sensitivity and specificity of the test by controlling the orientation of antibodies. The plate surface is coated with protein A followed by the trapping antibodies (polyclonal antibodies to angiotensinogen). Application of protein A increases the proportion of appropriately aligned antibody molecules. The plasma samples are then added, followed by the secondary antibody that is identical to the primary antibody (also polyclonal antibodies to angiotensinogen). The detecting agent is protein A conjugated to a marker enzyme (alkaline phosphatase). The protein A will only bind to the secondary antibody if the antibody is in the correct orientation. The substrate is added. Subjective analysis of ELISA results is achieved by quantifying the amount of light absorbed by the substrate. For p-nitrophenyl phosphate substrate, the substrate is exposed to light with a wavelength of 405nm and the absorbance is determined by measuring the amount of light that passes through the substrate. This is quantified as the absorbance value (O.D.) Method: Protein A was diluted 1 in 1000µl of coating buffer, 100µl of diluted protein A solution was added to each well of the ELISA plate. The plate was covered and incubated for 2 hours at 37ºC. The plate was washed vigorously with wash fluid PBS-Tween using a wash bottle. Each well was filled with PBS- Tween and left for three mins; the fluid was removed from the wells by snapping. The wash step was repeated three times and the plate was tapped dry on absorbent paper. Polyclonal angiotensinogen antibodies were diluted 1 in 1000µl of PBS- Tween, 100µl of diluted polyclonal anti AGT was added to each well. The plate was covered and incubated for 2 hours at 37ºC. The plate was removed and the wash procedure was repeated. Plasma samples were diluted 1 in 10µl in distilled water, 34 100µl of diluted sample was added to each well, and the plate was covered and kept in the fridge overnight. The plate was removed from the fridge and the wash procedure was repeated. Blank wells contained distilled water. Polyclonal angiotensinogen antibodies were diluted 1 in 1000µl of PBS- Tween, 100µl of diluted polyclonal anti AGT was again added to each well. The plate was covered and incubated for 2 hours at 37ºC (the antigen will be sandwiched between the antibodies). The plate was removed and the wash procedure was repeated. Protein A alkaline phosphatase conjugate was diluted 1 in 15000µl of conjugate buffer, 100µl of diluted conjugate was added to the wells of the ELISA plate. The plate was covered and incubated for 2 hours at 37ºC. The wash procedure was repeated. p-Nitrophenyl phosphate substrate was diluted 0.01g in 10000µl of substrate buffer; 100µl of diluted substrate was added to each well. The plate was covered and incubated in the dark for 1 hour at room temperature. The bottom of the plate was wiped with absorbent paper and inserted into the microplate reader. The absorbance values were measured at 405nm and recorded after 1hour, 3 hours and overnight. 3.16 Data Management. The Statistical Package for Social Sciences - SPSS for windows® Version 16.0 was used to statistically analyze the data obtained. Descriptive statistics was used to analyze all variables studied which include marital status, occupation, snack consumption, smoking practices, alcohol consumption and the genotypic frequencies of the four polymorphisms in the study population. Genotypic frequencies in control and hypertensive groups were compared by chi-square analysis. Continuous variables were compared between hypertensives and controls by independent t test. The effect of AGT genotype on BP was analyzed using general linear model. Influence of the gene variants on continuous variable was analyzed using one way ANOVA. Multiple regression analysis was also carried out using SBP or DBP as dependent variable, then sex, age, BMI and other variables were used as independent variables. P >0.05 was considered statistically significant. The linkage tables were plotted using Haploview version 4. CHAPTER FOUR RESULTS A sample population of 1224 individuals was genotyped to determine the frequencies of the M235T variant of the angiotensinogen gene, I/D allele of the ACE 1166 gene, A C of the AT1R gene and the C664G allele of the ANP gene and associate these alleles with hypertension status. Polymerase chain reaction and enzymatic digestion was performed on the 612 control and 612 patient samples collected from Uyo and Calabar to determine the frequency of the gene variants and its relationship with hypertension status. Protein A sandwich ELISA was also used to determine the concentration of angiotensinogen in the plasma samples of 300 patients and 300 controls in relation to the M235T allele of the AGT gene. 4.1 Agarose gel electrophoresis results After DNA extraction, the amount of DNA in the sample were quantified by checking the intensity of the bands on agarose gel and scored as faint, +, 2+ and 3+. Serial dilutions were done accordingly as 1 in 10, 1 in 50 and 1 in 100 dilution of T.E buffer for PCR. Plate 4.1 35 Angiotensinogen gene polymorphism: The normal individual M235M gives an undigested 165bp, a mutant individual M235T gives two fragments of 141bp and 24bp. Recessive individual T235T gives a 141bp fragment. However agarose gel allows the visualization of a 165bp fragment for M235M, a 141bp fragment for T235T, a 165bp and 141bp for the M235T individuals respectively. (Plate 4.2 and 4.3). Angiotensin converting enzyme gene polymorphism: Agarose gel allows the visualization of a 490bp band for a homozygous individual with the insertion (I) allele and a 190bp band for a homozygous individual with the deletion (D) allele. The heterozygous individual was identified by the presence of the 190bp and the 490bp PCR products (Plate 4.4). Angiotensin II type 1 receptor gene polymorphism: The Dde1 enzyme cuts the PCR product into two pieces, 600bp and 250bp in the A variant. An additional Dde1 recognition site is created in the C variant at nucleotide 1166 located within the 250bp fragment. The homozygous CC individual produces three bands (600bp, 140bp and 110bp long). The homozygous AA individual produces two bands (600bp and 250bp long). The heterozygous individual produces four bands 600bp, 250bp, 140bp and 110bp long (Lapierre et al., 2006).The homozygous CC individual was not observed (Plate 4.5 and 4.6). Atrial natriuretic peptide gene polymorphism: The Rsa1 enzyme cuts the PCR product into two pieces (134bp and 23bp). The common allele individual gives an undigested 157bp; the minor allele carrier gives two fragments of 134bp and157bp. Minor allele individual gives a 134bp fragment. However agarose gel does not allow the visualization of the 23bp fragment for a minor allele individual, (Kato et al, 2000). However, in the study population, only individuals with the 157bp product for the common allele were observed (Plate 4.7 and 4.8). 36 Plate 4.1 Gel electrophoresis showing DNA quality Legend: The amount of DNA was scored in Lanes 3,5,8,11 and 16 as 3+ Lanes 1,2,7 and 13 as 2+ Lanes 4,6,9,12,14 and 17 as + Lanes 10,15 and 18 as faint 37 Plate 4.2 Gel electrophoresis showing 165 bp PCR product after amplification of the angiotensinogen gene Legend: Lane M is the 1kb plus DNA ladder Lanes 1-16 contain the amplified PCR product 38 M 1 2 3 4 5 6 7 8 9 10 11 12 165 bp 141 bp Plate 4.3 Agarose gel electrophoresis showing the amplification of the 165 bp fragment after enzymatic digestion with the Tth 111l restriction endonuclease enzyme. Legend: Lane M is a 1kb plus DNA ladder Lane 1 is a 165 bp PCR product Lanes 2, 3, 6, 8, 9 and 10 are 141 bp digested fragments showing recessive homozygous individuals TT Lanes 4, 5, 7 and 11 are 165 and 141 bp fragments showing heterozygous individuals MT Lane 12 is a 165 bp undigested fragment showing a normal wild type MM 39 Plate 4.4 Agarose gel showing the amplification of the insertion/deletion of the angiotensin converting enzyme gene Legend: Lane M is the 100bp DNA ladder Lanes 1, 4, 5, 6, 7, 9, 10, 12, 13, 14,15,19 and 21 are the 490bp and 190bp PCR products showing heterozygous individuals I/D Lanes 2, 3, 8, 11, 16 and 20 are the 190bp PCR product showing individuals that have a homozygous deletion I/I Lane 18 is the 490bp PCR product showing an individual with homozygous insertion D/D Lane 17 did not amplify and the PCR had to be repeated 40 Plate 4.5 Agarose gel electrophoresis showing the 850bp PCR product of the Angiotensin II type I receptor gene. Legend Lane M is the 100bp DNA ladder Lane 1-19 are the amplified PCR product 41 Plate 4.6 Agarose gel electrophoresis showing digestion of the 850bp PCR product by the Dde1 restriction endonuclease. Legend Lane M is the 100bp DNA ladder Lane 1 is the PCR product Lanes 2, 3, 4, 5 and 7 are the homozygous AA individuals Lanes 6 and 7 are the heterozygous AC individuals Lane 8 was faint and the digestion was repeated 42 Plate 4.7 Agarose gel electrophoresis showing 157 bp PCR product of the atrial natriuretic peptide gene. Legend Lane M is the 100bp DNA ladder Lane 1-15 are the amplified PCR product 43 Plate 4.8 Agarose gel electrophoresis showing the 157bp product after enzymatic digestion with Rsa1 restriction endonuclease. Legend Lane M is the 100bp DNA ladder Lane 1-16 are the undigested product 4. 2 Demographic Data There were a total of 1,224 subjects recruited into the study, consisting of 612 hypertensives (225 males and 387 females) and 612 normotensives (272 males and 340 females) fig 4.1 and as a consequence more females were observed with TT for AGT and I/D and D/D genotype for the ACE, fig 4.2 - 4.5 shows the distribution of the polymorphisms by gender in the study population. The Efiks and the Ibibios (34.2; 32.4% respectively, n=612) were the main ethnic groups among the patients. The Ibibios (37.1%, n=612) were the predominant ethnic group among the controls. Fig 4.6-4.10 shows the ethnic groups and the distribution of the genes by ethnic group in the study population. The major ethnic groups had a higher frequency I/D variant among the controls and D/D genotype among the patients for the ACE. Most ethnic groups showed a high frequency of the TT genotype. Table 4.1 shows the genotype and allele frequencies of the polymorphisms among the major ethnic groups in Calabar and Uyo. 44 4.3 Genotypic frequencies The prevalence of AGT mutation was 88.4% for hypertensives and 92.2% for controls (homozygous mutation). 10.9% hypertensives and 7.5% control for the heterozygous mutation. The wild type allele was prevalent at 0.3% and 0.7% for patients and controls respectively. For the I/D allele of the ACE gene, the deletion was 45% and 39% (homozygous),the carriers of the deletion were 43% and 49% in the patient and control population, while the insertion allele was 12% in both control and patient populations. For the ATIR allele, 99% of the study population had the wild type allele and 1% was heterozygous carriers of the mutation. Only the wild type gene was observed for the ANP allele in the study population Table 4.2. Among the Efiks which are the predominant ethnic group in Calabar town, the genotype distribution of the M235T allele was 1, 16, 192 and 0, 10, 163 for the MM, MT, TT, genotypes among patients and control groups. The frequency of the 235T allele was 0.97. For the ACE polymorphism, the genotype frequency was 22, 91, 96 and 27, 78, 68 for the II, ID, DD genotypes among patients and controls. The frequency of the D allele was 0.70. For angiotensin 11 type 1 receptor gene, the genotype frequencies were 208, 1 and 174, 1 for the AA and AC genotypes. The CC genotype was not observed in this population. The frequency of the C allele is 0.003 in the controls and 0.002 in the patients. Among the Ibibios which also happens to be the predominant ethnic group in Uyo town. The genotype distribution of the M235T allele was 1, 24, 173 and 0, 16, 211 for the MM, MT, TT genotypes in patient and controls. The frequency of the 235T allele was 0.97. For the ACE polymorphism, the genotype frequencies were 22, 80, 96 and 30, 112, 85 for II, ID, DD frequencies with the D allele frequency as 0.70. For the angiotensin 11 type 1 receptor gene polymorphism, the genotype frequencies were 197, 1 and 286, 3 for the AA and AC genotypes, the CC genotype was also not observed in this group. The C allele frequency was 0.005 and 0.003 in the control and patient population Table 4.1. There were no significant differences between the genotype frequencies of hypertensive and the control groups by χ2 analysis for all the polymorphisms under consideration in this study. When continuous variables were compared between hypertensive and control groups, significant differences existed between the age, BMI, systolic and diastolic blood pressure of controls and patients. 4.4 Blood Pressure: For patients the mean diastolic blood pressure was 93.25 ±13.768, the mean systolic blood pressure was 161.14 ±23.247. For the controls, the mean systolic blood pressure was 116.76 ±9.19; the mean diastolic blood pressure was 72.181 ±8.41. Table 4.2 According to the JNC classification on hypertension, 265 patients had stage one hypertension and 347 patients had stage two hypertension, for the systolic BP measurement. From the diastolic BP measurement, 366 patients were grouped into the stage 1 category and 246 patients had stage 2 hypertension. For the systolic BP measurement in controls, 350 were classified into the prehypertension group while 262 were classified as normal. For the diastolic BP measurement, 279 controls were classified into the prehypertension group and 333 controls as normal. Table 4.3 45 Fig 4.1 Distribution by gender in the control and patient groups 46 Fig 4.2 Distribution of the AGT M235T polymorphism by gender among the patient and control population Legend: MM is the wild type individual MT is the heterozygous mutant individual TT is the homozygous recessive mutant individual 47 Fig 4.3 The ACE I/D allele distribution by gender in the control and patient population Legend: II is the wild type insertion individual ID is the heterozygous deletion individual DD is the homozygous recessive deletion individual 48 Fig 4.4 The ATIR allele distribution by gender in the control and patient population Legend: AA is the homozygous wild type individual AC is the heterozygous mutant individual CC is the homozygous recessive mutant individual 49 Fig 4.5 The ANP allele distribution by gender among the control and patient population Legend: CC is the wild type homozygous individual GC is the heterozygous mutant individual GG is the homozygous recessive individual 50 Fig 4.6 Ethnic distribution among the Control and Patient groups. 51 Fig 4.7 Distribution of the AGT M235T polymorphism among the Ethnic groups in the control population. Legend: MM are the homozygous dominant individuals MT are the heterozygous mutant individuals TT are the homozygous recessive individuals 52 Fig 4.8 Distribution of the AGT M235T polymorphism among the Ethnic groups in the Patient population. Legend: MM are the homozygous dominant individuals MT are the heterozygous mutant individuals TT are the homozygous recessive individuals 53 Fig 4.9 Distribution of the ACE I/D polymorphism among the Ethnic groups in the Control population. Legend: II are the homozygous insertion individuals ID are the heterozygous individuals DD are the homozygous deletion individuals 54 Fig 4.10 Distribution of the ACE I/D polymorphism among the Ethnic groups in the Patient population. Legend: II are the homozygous insertion individuals ID are the heterozygous individuals DD are the homozygous deletion individuals 55 56 Table 4.1 Genotype and allele frequencies of the polymorphisms among the major ethnic group in Calabar and Uyo. AGT ACE AT1R MM MT TT M T II ID DD I D AA AC CC A C Efiks Patients 1 16 192 0.04 0.96 22 91 96 0.3 0.7 208 1 0 0.998 0.002 Controls 0 10 163 0.03 0.97 27 78 68 0.4 0.6 174 1 0 0.997 0.003 Ibibios Patients 1 24 173 0.07 0.93 22 80 96 0.3 0.7 197 1 0 0.997 0.003 Controls 0 16 211 0.04 0.96 30 112 85 0.3 0.7 286 3 0 0.995 0.005 57 Table 4.2 Genotype and allele frequency of the RAAS and ANP polymorphisms in the patient and control population. Groups N Genotype Frequencies Allele Frequencies M235T MM MT TT M T Odds Ratio Hypertensives observed 612 4 67 541 75 1149 0.01 0.11 0.88 0.06 0.94 Hardy-Weinberg predicts expected 0.004 0.113 0.884 Controls observed 612 2 45 565 49 1175 0.003 0.075 0.922 0.04 0.96 Hardy-Weinberg predicts O.R=0.65,95%CI(0.396- expected 0.002 0.077 0.921 1.074) I/D II ID DD I D Hypertensives observed 612 73 262 277 408 816 0.33 0.12 0.43 0.45 0.67 Hardy-Weinberg predicts expected 0.11 0.44 0.45 Controls observed 612 74 303 235 451 773 0.37 0.122 0.490 0.388 0.63 Hardy-Weinberg predicts O.R=1.15,95%CI(0.923- expected 0.137 0.466 0.397 1.456) AT1R AA AC CC A C Hypertensive observed 612 605 7 0 1217 7 0.994 0.99 0.01 0 0.006 Hardy-Weinberg predicts 0.99 0.01 0 58 expected Controls observed 612 606 6 0 1218 6 0.99 0.99 0.01 0 0.01 Hardy-Weinberg predicts expected 0.98 0.02 0 ANP CC CG GG Hypertensives observed 612 612 0 0 Controls observed 612 612 0 0 59 Table 4.3 Classification of the study population according to the JNC V11 classification of blood pressure. Blood Groups according to Mean Blood Groups pressure No of JNC pressure Individuals Classification Patients Systolic 265 Stage 1 hypertension 347 Stage 2 hypertension 161.11 ± 23.26 Diastolic 366 Stage 1 hypertension 246 Stage 2 hypertension 93.25 ± 13.77 Control Systolic 350 Prehypertension 262 Normal 116.25 ± 9.22 Diastolic 279 Prehypertension 333 Normal 72.48 ± 8.37 60 4.5 Age: The hypertensive subjects ranged from 24 to 90 years old with a mean age of 51.2 years. Among the patient group, 463 (75.7) persons were more than 40 years of age and 149 (24.3) patients were less than 40 years. Normotensives ranged from 20 to 73 years old with a mean age of 31.9 years, 498 (81.3%) controls were less than 40 years of age while 114 (18.6%) controls were above forty years Fig 4.11. 4.6 Knowledge of and family history of hypertension: Among the hypertensive group, about 71% were fully aware of their hypertension status, 29% were not aware of their status Fig 4.12. 330 persons (53.9%) reported they do not have a positive history of hypertension.163 persons (26.6%) have a positive history of hypertension. 119 (19.4%) persons have no idea. None of the normotensives had hypertension although 22.7% had a positive family history of hypertension. 26.6% reported a negative family history of hypertension, 50.7% had no idea if there was any history of familial hypertension. 4.7 Smoking and alcohol consumption status: 603 (98.5%) of patients were non smokers, 594 (97.1%) of normotensives were non smokers. 434 (70.9%) of patients do not consume alcohol, 19.1% consume very little alcohol occasionally. 339 (55.4%) of controls do not consume alcohol, 201 (32.8) consume alcohol occasionally. 77 (11.8%) persons consumed alcohol on a regular basis. Figure 4.13-fig 4.16 4.8 Salt consumption: Salt intake was normal in 509 (83.2%) patients, 103 (16.8%) consume extra salt. controls whose salt intake was considered normal was 467(76%); the remaining 145 (24%) take more salt than normal. Fig 4.17- fig 4.19 61 Fig 4.11 Age distribution of patient and control groups 62 Fig 4.12 Knowledge of hypertension status among patients Legend 71% were aware of their hypertension status 63 Fig 4.13 Smoking status among control and patient group 64 Fig 4.14 Alcohol consumption among control and patient groups Legend: Individuals under no alcohol response do not consume alcohol Individuals under the occasional drinkers responses consume alcohol once in a while Individuals under the regular drinkers responses are regular consumers of alcohol 65 Fig 4.15 Distribution of the AGT M235T polymorphism and alcohol consumption in the study population. Legend: MM are the homozygous dominant individuals MT are the heterozygous mutant individuals TT are the homozygous recessive individuals 66 Fig 4.16 Distribution of the ACE I/D polymorphism and alcohol consumption in the study population. Legend: II are the homozygous insertion individuals ID are the heterozygous individuals DD are the homozygous deletion individuals 67 Fig 4.17 Salt intake among control and patient groups Legend: No extra means subjects do not add extra salt to their diet Extra means subjects add extra salt to their diet 68 Fig 4.18 Distribution of the AGT M235T polymorphism and salt intake among the study population. Legend: The TT individuals that consume no extra salt were the predominant group among the patients and the controls. MM are the homozygous dominant individuals MT are the heterozygous mutant individuals TT are the homozygous recessive individuals 69 Fig 4.19 Distribution of the ACE I/D polymorphism and salt intake among the study population. Legend: The I/D individuals that consume no extra salt were the predominant group among the controls and the D/D were predominant among the patients. II are the homozygous insertion individuals ID are the heterozygous individuals DD are the homozygous deletion individuals 70 4.9 Educational Attainment Among the patient group, 245(40%) individuals attended only primary school, 167(27.3%) individuals attended secondary, 101(16.5) individuals attended tertiary institution and 99(16.2) individuals had no form of formal education. Among the control group, individuals who attended primary schools were 139(22.7%); secondary schools were 246(40.2%); tertiary institutions were 201(32.8%) and individuals that had no formal education were 26(4.2%) Fig 4.20- 4.22. 4.10 Body mass index 2 In the patient population, BMI below 24.9kg/m was observed in 234 persons 2 (31.54%), BMI between 25 – 30kg/m was observed in 193 persons (38.23%) and 2 BMI above 30kg/m was found in 185 persons (30.23%). In the controls, BMI above 2 2 30kg/m was found in 80 persons (12.79%), BMI between 25 – 30kg/m was found in 2 121 persons (20%) and a BMI below 24.9kg/m was observed in 411 persons (67.10). Fig 4.23 4.11 Exercise 33.2% controls do not carry out any form of exercise, 296(48.4%) controls indicated strolls to be the main exercise usually carried out. In the patient group, 293(47.9) do not carry out any form of exercise, 281(45.9) indicated strolls to be the main form of exercise Fig 4.24-Fig 4.27. 4.12 Marital status In the control group, 294 (48%) persons were married and 316 (51.6%) persons were singles. For the patients, 523 (85.5%) persons were married and 54 (8.8%) were singles. There was no divorcee among controls but 1 among patients. There were 2 (0.3) widows among controls and 34 (5.5) widows among patients Fig 4.28. 4.13 Visits to fast food joints 547 patients do not go to fast food joints at all except for 65 persons. 45.9% mentioned strolling as their exercise type, 47.9% of patients did not practice in any form of exercise Fig 4.29. 71 Fig 4.20 Educational levels among patient and control groups. 72 Fig 4.21 Distribution of the AGT M235T polymorphism and educational levels among the study population. Legend: MM are the homozygous dominant individuals MT are the heterozygous mutant individuals TT are the homozygous recessive individuals 73 Fig 4.22 Distribution of the ACE I/D polymorphism and educational levels among the study population. Legend: II are the homozygous insertion individuals ID are the heterozygous individuals DD are the homozygous deletion individuals 74 Fig 4.23 Body Mass Index observed among control and patient groups. 75 Fig 4.24 Exercise types observed among control group 76 Fig 4.25 Exercise types observed among patient group 77 Fig 4.26 Distribution of the ACE I/D polymorphism and exercise types among the study population. Legend: II are the homozygous insertion individuals ID are the heterozygous individuals DD are the homozygous deletion individuals 78 Fig 4.27 Distribution of the AGT M235T polymorphism and exercise types among the study population. Legend: MM are the homozygous dominant individuals MT are the heterozygous mutant individuals TT are the homozygous recessive individuals 79 Fig 4.28 Marital status of control and patient groups 80 Fig 4.29 Frequency of visits to fast food joints in the control and patient group Legend: Individuals who give a yes response visit the fast food joints at least once a week Individuals who give a no response do not go to the fast food joints at all 81 4.14 Occupation Among the patients, the main occupational groups were Pensioners13.6%, Self employed/Business 13.9%, Civil/Public servant 11.4%, Farmers 10%, Traders 7%. Among the controls, the main occupational groups were Academics 29.7%, Self employed/Business 17.2%, Traders 11.9, Civil/Public servant 11.9%, Domestic workers 11.8%. Fig 4.26 -fig 4.29 4.15 Regression analysis By multiple regression analysis, age was the predictor for SBP and DBP in the hypertensive group r=0.604 SBP, r=0.594 DBP, p ≤ 0.05. Age was a predictor for DBP in the control group r=0.542, gender was a predictor for SBP in the control group. Gender, body mass index, AGT genotype, ANP genotype, ACE genotype and other independent variables were not predictors for SBP and DBP in the hypertensive group. The influence of AGT genotype on continuous variable was compared using 1 way ANOVA. There was significant differences in the age and systolic in the control group and the systolic in the patient group but no significance differences between the continuous variation of other groups. When blood pressure and other variables using general linear model ANOVA with no adjustments age was significant for the MT and TT variables. See tables in appendix. 4.16 Hardy-Weinberg Theory The observed frequencies of the polymorphism are not equal to those frequencies predicted by hardy-Weinberg which means there are evolutionary mechanisms influencing the loci under consideration. Thus the population does not meet the assumption of the hardy-Weinberg theory Table 4.2. 4.17 Measurement of plasma angiotensinogen using sandwich ELISA In the control group, the mean O.D value for plasma angiotensinogen was 0.53 in individuals with the M235T variant; 0.49 in individuals with the T235T variant. There was only one individual with the M235M variant (O.D value 0.28). In the patients, the mean O.D value was 0.71 in individuals with the M235T variant; 0.66 in individuals with the T235T variant. There was also only one individual with the M235M variant (O.D value 0.41) among the patient group. Several dilutions of the purified protein did not yield any results, thus the actual concentration of the protein in the samples could not be determined Table 4.4. 4.18 Analysis of linkage disequilibrium Linkage disequilibrium (LD) was calculated between all possible pairs of the four polymorphisms. The was Linkage disequilibrium between the polymorphisms of 2 the C664G and the M235T D'=1.00; r = 0.86 in the control group Table 4.5. In the patient group, Linkage was also observed between C664G and I/D polymorphism 2 D'=0.99; r = 0.39 Table 4.6. All other comparison between alleles of the polymorphisms have negative correlation values. 82 Fig 4.30 Occupational groupings for patient population 83 Fig 4.31 Occupational grouping for control population. 84 Fig 4.32 Distribution of the AGT M235T polymorphism and occupation among the study population Legend: MM are the homozygous dominant individuals MT are the heterozygous mutant individuals TT are the homozygous recessive individuals 85 Fig 4.33 Distribution of the ACE I/D polymorphism and occupation among the study population Legend: II are the homozygous insertion individuals ID are the heterozygous individuals DD are the homozygous deletion individuals 86 Table 4.4 Characteristics of the individuals used in the protein A sandwich ELISA for the measurement of plasma angiotensinogen Controls Patients O.D values Ihr 0.16 ± 0.06 0.17 ± 0.05 for ELISA 3Hrs 0.26 ± 0.12 0.38 ± 0.20 Overnight 0.50 ± 0.21 0.66 ± 0.33 Genotype TT 0.49 ± 0.20 0.66 ± 0.33 MT 0.53 ± 0.27 0.71 ± 0.33 MM 0.28 0.41 Age 34.56 ± 10.27 53.45 ± 14.14 BMI 23.05 ± 6.51 26.31 ± 5.78 Systolic Blood pressure 115.60 ± 9.63 163 ± 23.87 Diastolic blood pressure 71.39 ± 8.50 94.87 ± 13.59 87 Table 4.5 Linkage disequilibrium matrix for controls Multi- Multi- Multi- Multi- Mutual Allelic Allelic Allelic Allelic ChiSquare ChiSquare ChiSquare Row Marker1 Marker2 Information D D' R RSquare df value p-value 1 C664G A1166C 0.0000 0.0000 0.0000 0.0000 0.0000 1 0.0 1.0000 - 2 C664G I_D 0.3461 0.1258 1.0000 0.5799 0.3362 1 411.6 0.0000 3 C664G M235T 0.8051 0.2312 1.0000 0.9275 0.8602 1 1052.9 0.0000 - 4 A1166C I_D 0.3380 0.1240 1.0000 0.5714 0.3265 1 399.6 0.0000 - 5 A1166C M235T 0.7786 0.2278 1.0000 0.9139 0.8352 1 1022.3 0.0000 - 6 I_D M235T 0.3071 0.1164 1.0000 0.5378 0.2892 1 354.0 0.0000 88 Table 4.6 Linkage disequilibrium matrix for patients Multi- Multi- Multi- Multi- Mutual Allelic Allelic Allelic Allelic ChiSquare ChiSquare ChiSquare Row Marker1 Marker2 Information D D' R RSquare df value p-value - 1 C664G A1166C 0.9549 0.2471 1.0000 0.9886 0.9774 1 1196.3166 0.0000 1 C664G I_D 0.3836 0.1420 0.9900 0.6278 0.3941 1 482.3452 0.0000 1 C664G M235T 0.0000 0.0000 0.0000 0.0000 0.0000 1 0.0000 1.0000 - 2 A1166C I_D 0.3759 0.1403 0.9897 0.6204 0.3849 1 471.1052 0.0000 - 1 A1166C M235T 0.7323 0.2208 1.0000 0.8885 0.7894 1 964.6408 0.0000 - 3 I_D M235T 0.3175 0.1262 0.9862 0.5615 0.3153 1 385.3202 0.0000 89 CHAPTER FIVE DISCUSSION Genetic variations of genes encoding components of the renin-angiotensin- aldosterone-systems (RAAS) have been associated with susceptibility to hypertension making them strong candidate genes for investigating the genetic basis of hypertension. In addition to the RAAS, the natriuretic peptide system also affects blood pressure directly through its vasodilatory and natriuretic activities and indirectly by inhibiting the RAAS. This has also generated interest in the role of ANP in the development of hypertension. The renin angiotensin aldosterone system plays a major role in blood pressure regulation and angiotensinogen (AGT), a key substrate in this pathway has been an attractive candidate gene for the study of hypertension by many investigators (Inoue et al., 1997; Jain et al., 2002; Markovic et al., 2005). The T235 mutation was over 90% prevalent in the study population (0.94 for hypertensives and 0.96 for controls) which was very high, this is consistent with literature. Nigerians (specifically among the Yorubas) have been reported to have a high frequency of this molecular variant, between 80-93%. The frequency of this allele is also high in Africans and African Americans, about 0.92 (Rotimi et al., 1994; 1997). The frequency of the T235 mutation among hypertensive Malaysians was reported as 0.45 and 0.75 among the Japanese (Say et al., 2005; Nishiuma et al., 1995). In the present study, it was found that the frequency of the M235T polymorphism of the AGT gene though very high was not associated with hypertension, the odds ratio for hypertension in this study was 0.65 (95% CI, 0.396 to 1.074) which means in effect that the M234T allele is not a positive predictor for hypertension in the study population. An explanation for this observation could be differences in genetic background. The participants were not selected according to family history of hypertension. A more heterogenous family background could dilute the genetic component and emphasize the importance of enviromental factors. Results of association studies between angiotensinogen and hypertension have been conflicting; some studies have indicated that the M to T amino acid substitution at position 235 was associated with hypertension in several ethnic groups including Caucasian and Japanese populations (Hata et al., 1994; Matinez et al., 2002, Pereira et al., 2003). Jeunemaitre et al. (1992) were the first to report the linkage of the molecular variant M235T with hypertension in Whites/Caucasians. Subsequent studies among Whites/Caucasians supported an association (Schmidt et al., 1997). Early Chinese and Taiwanese studies had also reported positive associations with hypertension (Niu et al., 1999; Chiang et al., 1997). Studies in the Japanese population found a positive association between hypertension and the M235T variant with an odds ratio of 2.67 (Hata et al., 1994; Nishiuma et al., 1995). Markovic et al. (2005) reported a significant association of nucleotide base substitutions at position 6, 29, 793, and 776 in the promoter region of the angiotensinogen gene with hypertension in African-Americans and Caucasians. Kunz et al. (1997) in a meta analysis performed to examine the association of the 235T allele and hypertension in 5493 White patients showed an odds ratio of 1.20 (95% CI, 1.11 to 1.29), the odds increased to 1.42 (95% CI, 1.25 to 1.61) in subjects with a positive history of hypertension. Pereira et al. (2003) reported an odds ratio of 1.33 (95% CI, 1-04 to 1.70) for hypertension in individuals with the TT genotype. 90 Most females (both in controls and patients) were of the T235T genotype. A study had indicated that homozygosity for the Thr 235 allele predicted risk for hypertension in women and not in men (Sethi et al., 2001). Other studies provided no evidence for association with essential hypertension while stratifying for sex (Hegele et al., 1994; Fornage et al., 1995). Periera et al. (2003) also reported no association between gender and T allele in a cross sectional study involving 647 females and 776 males though the study was not confined to essential hypertensive patients only. Lindan et al. (2010) observed a significant association of the TT genotype of the AGT polymorphism with essential hypertension in Han Chinese, an odds ratio of 1.54 (95% CI, 1.16 -2.03). Say et al. (2005) reported a high odds ratio for the TT genotype of the AGT allele with hypertension among Malaysian subjects 1.98 (95% CI, 1.46 to 2.67). The T235T variant was also reported to be associated with preeclampsia (Ward et al., 1993; Procopciuc et al., 2002). In Han Chinese women, maternal AGT allele was reported to have no effect on the risk of pregnancy-induced hypertension (PIH) but fetal 235T allele was significantly associated with PIH in the women. Fetuses with the TT genotype have a protective effect against PIH in the study population (Xiang et al, 2011). Other studies on White/Caucasians did not support any association of the M235T variant with hypertension (Fornage et al., 1995; Hegele et al., 1997). Some Japanese studies did not find any association between the M235T and hypertension (Morise et al., 1995; Iwai et al., 1995). Association studies in Africans and African- Americans also found a negative association( Rotimi et al., 1994; Caulfield et al., 1995; Wu et al., 2003). The mechanism by which the molecular variant M235T allele of the AGT gene is related to hypertension is poorly understood. Although the AGT 235T allele was found to be in complete linkage disequilibrium with a guanine to adenosine transition at -6bp upstream of the initiation site of transcription (Inoue et al., 1997). In vitro test of promoter activity and DNA binding studies with nuclear proteins show that this nucleotide substitution affects the basal transcription rate of the gene in various cell lines thereby affecting the AGT T235 variant, increased plasma AGT levels that might lead to increased blood pressure (Jeunemaitre et al., 1992c). The T allele was associated with increased plasma AGT (Bloem et al., 1997, Rotimi et al., 1997). Elevation in plasma levels of angiotensinogen has been associated with hypertension. Well developed assays to measure this protein are few and expensive. Previous research carried out usually measure renin using the Plasma Renin Activity (PRA) test involving a radioimmuno precipitation method. The present study used a sandwich ELISA method to assess the levels of plasma angiotensinogen in the participants. The mean O.D values for plasma angiotensinogen was significantly higher in the patients than the controls with the mutant T allele implying an association with hypertension. There is need to further improve this technique which might be a useful tool in measuring angiotensinogen for diagnostic purposes. The protein A sandwich ELISA was sensitive enough to identify the presence of the angiotensinogen in the plasma of patients and controls, the O.D values indicate the presence of higher concentration of angiotensinogen in the patients than in the control which is in line with literature (Jeunemaitre et al., 1992 and Corvol et al., 1999). Corvol and Jeunemaitre (1997) reported that the M235T allele was associated with a 10-30% increase in plasma angiotensinogen which is able to increase blood pressure, 91 thus facilitating hypertension. The limitation of this study was the fact that the assay could not pick up the standard angiotensinogen dilutions to enable a proper interpretation of the concentration of angiotensinogen in each plasma sample. More work still needs to be carried out in this area. The ACE genotype frequencies were 73(12%), 262(43%) and 277(45%) for the II, ID, DD respectively in the patient group. The ACE alleles were 85(12%), 341(49%), 270(45%) for the II, ID, DD respectively in the control group. A higher frequency of the ID allele was observed in controls of which 208(61%) were females. Among the major ethnic groups residing in the two towns, the D allele frequency was 70% and the I allele was 30% which was high compared to literature. Rotimi et al. (1997) reported the frequency of the D allele among African Americans as 63% while Morshed et al. (2002) reported 69.3% for the D allele in hypertensives and 45.7% in controls. He also observed a higher frequency of the I allele in the controls (54.2%) than the hypertensives (50%). Wang et al. (2005) reported the D allele frequency as 40.8% which is lower than what was obtained by O‟Donnel et al. (1998) in European samples (55.3%). Kario et al. (1996) reported a frequency of 34% for Japanese individuals. Dankova et al. (2009) reported 0.53 frequency for the mutant D allele in Slovak subjects and 0.447 in Romany subjects. A frequency of 52.9% for patients and 56.3% for controls was reported for the D allele in a Lebanese diabetic cohort by Chmaisse et al. (2009). Ismail et al. (2004) reported a significantly higher frequency (0.55) of the ACE II genotype in the hypertensive group than in the control group of the same age but no overall significant differences were observed between the II, ID, DD ACE genotypes. The D allele has been associated with hypertension in some studies in White American and Japanese men but not in women (O‟Donnel et al., 1998; Katsuya et al., 1998). Sagnella et al. (1999) however observed a significant association between the D allele and hypertension in women of African descent. Many studies have failed to establish an association between the D allele and hypertension (Hsieh et al., 2000). A strong association of the I allele was found in an Australian population with familial hypertension (Zee et al., 1992). The conflicting results of I/D polymorphism of the ACE gene in hypertension has been attributed to gender and ethnic differences (Ramachandran et al., 2008). In this study, the I/D allele of the ACE gene is associated with an increased risk for hypertension with an odds ratio of 1.15(95% CI, 0.924 -1.456). Ji et al. (2010) observed a higher odds ratio of 1.61(95% CI, 1.32 – 1.98) for the ACE gene among the Han Chinese population. Sagnella et al. (1999) reported an odds ratio of 1.65 (95% CI, 1.04- 2.64) in women of African descent (OR=2.54; 95%CI, 1.38 -4.65) but not in men of African descent (OR=0.79; 95% CI, 0.36 – 1.72). Bhavani et al. (2004) reported a significant association of the ACE I/D allele with hypertension in men with age adjusted odds ratio of 2.25 (95% CI, 1.14 – 4.42) and 2.20 (95% CI, 1.22 – 3.80) for DD and ID respectively. In women there was no significant association of ACE genotype with hypertension, age adjusted odds ratio being 1.20 (95% CI, 0.38 – 3.92) and 0.44 (95% CI, 0.17 – 1.06) respectively for the DD and ID genotypes. Das et al. (2008) observed that the odds of being hypertensive in a population of Asian Indians of Calcutta was 7.48 (95%CI, 1.75 – 30.190) in the DD homozygous individual suggesting a very strong association of the ACE polymorphism with essential hypertension in Asian Indians. Sameer et al. (2010) observed a strong association between the ACE polymorphism and hypertension among the peoples of Kashmir, India. 92 World distribution of the D allele according to Salem (2008) suggest that the I/D polymorphism in the human ACE gene is of African origin. The allele is believed to have moved out of Africa with Paleolithic (second part of the stone age that began about 750,000 to 500,000 BC and lasted until the end of the ice age about 8,500BC) migrations 100.000 years ago. The ACE I/D polymorphism is due to an insertion of a 287bp AluYa5 element into intron 16 of the gene (Rigat et al., 1992). This insertion is believed to have occurred a few million years ago during the evolution of primates (Jurka, 2004). Although an insertion or a deletion event is implied in the I/D polymorphism, only an insertion event occurred. This makes the D allele without an insertion the ancestral state of the gene. Primate specific Alu elements have been reported to be the most abundant transposable elements in the human genome making up more than 10% (Batzer and Deininger, 2002). The mechanism by which D allele leads to blood pressure elevation is not clearly documented in literature. It has been observed that there were differences in the response of different ethnic groups to ACE blockers and diuretics in the treatment of hypertension (Douglas et al., 2003; Wright et al., 2005). Whites were observed to have better response to ACE blockers than Blacks (those of African ancestry) while the reverse was the case with diuretics ACE blockers or inhibitors are a type of drug commonly used to treat hypertension (Gard, 2010). These drugs reduce the activity of angiotensin converting enzyme, thus reducing the concentration or levels of angiotensin II facilitating a reduction in hypertension (Johnson et al., 2008). Exner et al. (2001) found in a one year therapy with the ACE inhibitor enalapril, that it was associated with significant reductions in blood pressure in White patients but not among black patients, using cardiovascular consequences such as fatal and non fatal myocardial infarction and heart failure as end points. The D allele of the ACE I/D polymorphism is associated with increased ACE activity which is associated with increased incidence of cardiovascular disease and a resistance to ACE inhibitor therapy. The molecular mechanisms are not clearly explained in literature (Wright et al., 2005). In Caucasians and Asians, the ACE polymorphism has been observed to produce higher levels of protein in the blood, meaning that if these patients require treatment for high blood pressure, they should not respond to ACE inhibitors. However, African Americans have the same gene variant but this does not increase the blood protein levels in this population, thus ACE inhibitors should work in this population. This is conflicting with what is generally reported among blacks. The reason for this difference is unclear (Mellen and Herring, 2005). Mc Donagh et al. (2011) further explained that the plasma soluble form of ACE is not involved in the cleavage of angiotensin I to angiotensin II and that ACE inhibitors usually target tissue bound ACE that are actually involve in angiotensin I conversion thereby resulting in the down regulation of angiotensin II that subsequently lowers blood pressure. However Gainer et al, (2001) reported that bradykinin which is a potent vasodilator contributes to the effect of angiotensin- converting enzyme ACE inhibition in humans. Decreased production of bradykinin or decreased vasodilation in response to bradykinin is thought to play a role in hypertension and also in the decreased antihypertensive response to ACE inhibition in Blacks (Gainer et al., 2001) The CC allele of the AT1R gene was not observed in this study population. 99% of the population had the AA wild type gene. This is in contrast to what is reported in some literature. A high prevalence of the CC genotype was observed in Chinese hypertensives than controls (Ono et al., 2003). In a sample of Swedish twins, Iliadou et al. (2002) did not observe any association between the ACE I/D 93 1166 polymorphism or AT1R A C polymorphism and blood pressure. Schmidt et al. (1997) also did not detect any association between the A1166C allele and hypertension but a decreased prevalence of C allele was observed among hypertensives. Tiret et al. (1998) reported a higher prevalence of the C allele among female hypertensives than controls but no such observation among men. A higher prevalence of the CC genotype was observed in Chinese hypertensives than controls (Jiang et al., 2001). Generally large interethnic differences in the frequencies of genotype polymorphisms of the RAS exist in different populations. To explain ethnic differences observed in allele frequency in different study populations, Lynch et al. (2008) reported that there are differences in linkage disequilibrium for different loci among various populations, if such differences exist in regions where these variants are found and such variants are not causal but are in linkage disequilibrium with the putative locus, this might explain the inconsistency in results across population. Among the Efiks and the Ibibios, the major ethnic group in this study population, the frequency of the A allele was 99.7 and 0.3 for C allele, the average C allele frequency reported in the Chinese population is 0.11. In another study, the homozygous A1166 allele frequency was 92.8 among the studied subjects. The frequency of homozygous A allele was significantly higher in the hypertensives than the normotensive subjects (97.5% and 80% respectively) with a higher frequency among male patients (Farrag et al., 2011). Farrag et al. (2011) proposed that the A allele may be a predisposing factor for essential hypertension in Egyptians. Zhenni et al. (2001) observed only two genotypes AA and AC of the AT1IR polymorphism, but reported a higher frequency of the A allele among the patients than the controls. Lee and Kim (2003) observed 96% and 6% for the A and C allele of the angiotensin 11 type 1 receptor polymorphism in Korea. Hahntow et al. (2010) found the A allele to be associated with high blood pressure. They also reported that the A allele showing an association was not totally out of place given the fact that this locus had no major impact on hypertension phenotype as suggested by genome wide studies (Wu et al., 2006; Rice et al., 2006 and Newton-Cheh et al., 2009). Other studies (Stankovic et al., 2003; Ono et al., 2003) have reported the C allele instead to be associated with hypertension. The -C664G mutant of the atrial natriuretic peptide gene was not observed in this population. The wild type C664C allele of this polymorphism was present in both patient and control groups. This implies that this mutation has not been introduced into this population. however Rubattu et al. (2006) observed a 97.4% of the C664C allele and 2.6% of the C664G allele among hypertensive patients in Milan, Spain. Though the C664C allele frequency was high, the C664G allele was associated with left ventricular mass index in hypertension. Rubattu et al. (2007) also found that young men heterozygous for the G allele had an increased risk for an early onset of hypertension. The C664G, G1837A AND T2238C of the ANP gene has been investigated in association with hypertension and found to be monomorphic among the Chinese (Xue et al., 2008). More research needs to be carried out to confirm these results. Among the ethnic groups of the sample population, the Efiks were 384 persons and the Ibibio‟s were 487 persons, the Efiks and Ibibios made up the main ethnic groups in this study as is logical since the study was conducted in Calabar and Uyo. Most patients were married and advanced in age (above 40 years). Increase in age is thought to increase blood pressure because the arteries become hardened, less 94 active, kidney function decreases and the body does not process salt as well as before (Lloyd-Jones et al., 2005). It was observed that most individuals below the age of 40 years were normotensives. Awareness of hypertension status was about 70%. The remaining 30% consisted of individuals who were first timers at the clinic; individuals from the population whose blood pressure readings were discovered to have increased and had to register at the hypertension clinic for proper follow up. Tobacco smoking has been shown to increase blood pressure, blood pressure was observed to decrease in smokers who did not smoke for a week (John et al., 2006). Smoking and alcohol consumption was low in both populations. The hypertensive had been educated by their doctors not to consume alcohol. The reason for abstinence among controls was due mainly to their religious beliefs. The possibility exist that participants did not give a truthful answer because drinking is culturally frowned at. Strolls were the most frequent exercise on a regular basis reportedly carried out by both patients and controls, though 47.9% of patients did not perform any form of exercise (this group was made up mainly of the very elderly, above 60 years of age). Lack of exercise makes it easier to become overweight and increases the chances of high blood pressure. Exercise is an important treatment for hypertension, only people with severe uncontrolled hypertension are advised not to carry out any exercise. People whose hypertension are less severe or controlled by drugs are advised to participate in exercise as a way of managing their blood pressure. Salt intake was considered „normal‟ for persons who do not consume raw salt except for the quantity used in food preparation. Those classified into high salt intake group actually consume raw salt that is use it to drink garri, eat dry fish and add extra salt to their food when they feel the salt content is not enough. Salt metabolism and its effect on hypertension are extremely complex. Primary or essential hypertension and age-related increases in blood are almost absent in populations where individual consumption of salt (sodium chloride) is less than 50mmol per day but mainly observed in population in which people consume more than a 100mmol of salt per day (Kaplan, 2006). Although individual salt intake in most populations throughout the world exceeds 100mmol per day, most people remain normotensive. This implies that salt intake that exceeds 50 - 100mmol per day is necessary but not sufficient for an individual to develop the disease, other factors come into play. Weinberger et al. (2001) carried out a study and concluded that it is not the hypertension produced by salt that is the most important cause of health problems; instead it is whether the individual is salt sensitive (salt sensitivity is an increase in blood pressure in response to a higher salt intake than that in a baseline diet (Morris et al., 1999). For salt-sensitive individuals, the risk of dying from cardiovascular problems is increased with high dietary salt whether they are hypertensive or not. Iwamoto et al. (2004) explained how excess salt affects high blood pressure. The key substance is a hormone ouabain secreted by the adrenals which in turn affects two proteins that together regulate the sodium-calcium content of the smooth muscle cells of arteries. Excess salt intake stimulates the secretion of excess ouabain; this upsets the balance by disabling the sodium pump and causes sodium to accumulate in the artery cell. The excess sodium causes the Na - Ca2+ exchanger protein to bring in more calcium to replace the sodium and this in turn triggers artery constriction and hypertension. Another explanation for salt as predisposing factor for hypertension is that the human kidneys usually retains sodium and excrete potassium. Prehistoric 95 human who consumed a poor sodium and potassium rich diet were favoured by this mechanism. In this case sodium excretion is negligible and potassium excretion is high thus the need for potassium rich foods. This mechanism does not favour our modern day sodium- rich and potassium- poor diet. The end result of the failure of the kidney to adapt to this diet is an excess of sodium and a deficit of potassium in hypertensive patients. (Adrogué and Madias, 2007). Fast foods have become popular in the western world due to their taste, convenience and affordability. Fast foods have gained popularity among Nigerians though it is common among the rich because it is expensive. Fast foods contain high amount of saturated fats, trans fats, salt and in some cases sugars. Chronic consumptions of high amounts of fast foods for a long time increases a person's risk for adverse health conditions such as hypertension, atherosclerosis and heart failure. In this study, patients (89%) did not go to fast food joints, the reason being that doctors had advised against it. Even among controls, visit to fast food joints was still very low. The people of these areas still prefer to eat their local dishes of vegetables, root tuber and fresh sea foods. But this trend is likely to change in the near future with more fast food joints opening up and the people, youths to be precise, embracing the western way of living. A higher patient number do not consume snacks because they were advised by the doctors in the clinics to reduce intake of snacks for health reasons. Most controls in the study had a BMI less than or equal to 67.10%. In the patient population, overweight 38.23% and obesity 30.23% reach a moderate prevalence among patients. Hypertension has been reported to be strongly correlated with BMI. Weight gain in adulthood is seen as an important risk factor for hypertension (Jafar et al., 2006). Humayan et al. (2009) observed a high trend of hypertension with increasing BMI among Pakistanis, with a high incidence among 2 females whose weight was above normal that is less than 24.9 kg/m . Positive associations between body mass index and blood pressure have also been documented in cross sectional studies in different Asian population. Ethnic differences existed in the association between BMI and hypertension and in optimal mi cutoffs for overweight Chinese, Indonesians and Vietnamese adults. (Stamler et al., 1978; Stevens et al., 2002; 2008; Bell et al., 2002; Tuan et al., 2009) All the hypertensinogenic factors considered did not contribute significantly to the disease when regression analysis was carried out. Potential risk factors for hypertension are not only genetic variables but also psychological and environmental influences. Hypertension is therefore the end result of various events that develop gradually over many years. Studies have demonstrated a negative association of cardiovascular disease mortality and morbidity as measured by education and or occupation. These studies indicate that individuals with lower socioeconomic status were more likely to have cardiovascular diseases (Vargas, 2000). Socioeconomic differences play a significant role in the health status of a population and are likely to influence the pathogenesis of hypertension and access to preventive health services. It has been demonstrated that education may be the most judicious measure of socio economic status for epidemiological studies. Education plays an important role in guarding against disease influenced by one‟s lifestyle (Liberatos et al., 1988; Vargas, 2000). Hypertension, diabetes are common causes of cardiac, cerebrovascular and renal diseases, they are easily diagnosed and can be treated effectively to reduce death. Tedesco et al. (2001) reported that low knowledge of hypertension and its risk factor among the uneducated, made symptomless patients unwilling to alter their lifestyle, take 96 medication and visit health facilities when necessary to forestall some poorly perceived danger while the educated subjects are more likely to consider the health care need as a priority. In this study, the primary level of education had the highest number of hypertensives (40%), followed by the secondary level (27%). Those with no formal education were the least with 16.2%.This results suggest an association between low levels of education and the development of hypertension thus highlighting the need for increased awareness campaigns to enlighten the less educated to make them aware of the disease and the health facilities available to them. Adedoyin et al. (2005) reported low socio-economic status to have an inversely significant effect on systolic, heart rate and pulse rate thus implicating socio economic status in the development of hypertension among sedentary Nigerian adults. Population geneticist study frequencies of genotypes and alleles within populations, by comparing these frequencies with those predicted by null models that assume no evolutionary mechanisms are acting within the population; they draw conclusions regarding the evolutionary forces in operation. The hardy-Weinberg Equilibrium law serves as the basis null model for population genetics. Populations will confirm to the Hardy-Weinberg law only if no evolutionary forces influence the loci under consideration. These evolutionary forces include large population size where there is no genetic drift. Chance can alter allele frequencies through mating processes and death within small populations. Random mating where choice of mates by individuals is by chance and not influence by the genotype of the individuals in question. In natural population matings are random with respect to certain characteristics such as blood group but for some other characters such as tribes, matings are not random. This will no doubt affect the distribution of allele frequencies. There are also differences in the mutation rates between alleles at the same locus with natural selection favouring some alleles in some population. Reproductive isolation from other populations which means that there is no gene flow or migration into the population. With our efficient systems of transportation, migration has introduced a lot of genes into populations where they were not found initially. There should be no differential survival or reproduction among phenotypes that is natural selection does not act on the individuals (Wigginton et al., 2005). Due to selection, some phenotypes have been selectively favoured to suit different environments and of course reproduction will be more common among these phenotypes. The population under study does not conform to the hardy-Weinberg equilibrium theory which means some of these evolutionary forces are acting on the loci under consideration. There was linkage disequilibrium between the C664G and M235T polymorphisms in the controls and between C664G and I/D alleles in the patients. LD implies correlation between loci. This could mean that the alleles of the C664G and I/D polymorphism belong to the same haplotype block. In otherwords they are inherited together more often than chance would dictate. LD throughout a particular genome reflects the population‟s history, breeding systems and the pattern of geographic subdivision. While LD in a genomic region reflects the history of natural selection, gene conversion, mutation and other forces that could cause gene-frequency evolution (Slatkin, 2008). Since the study population was not a completely homogenous population from a particular area, these associations need further investigations in a more homogenous population. The results obtained from this study will form baseline information for these areas but more work still needs to be carried out to confirm these genotypes in a larger population. 97 Other genes like the Corin, fumin and genes that regulate other pathways outside the RAAS have also yielded results that have contributed to the molecular understanding of the molecular basis of hypertension but there still some polymorphism that act in linkage disequilibrium with the already identified gene to produce disease. These genes need to be identified to properly explain the molecular basis of hypertension. Candidate gene analysis and Linkage studies are limited in its explanation for the molecular basis of hypertension due to conflicting results from various studies but with the advent of Genome wide Association Studies (GWAS), significant strides have been made in our understanding and knowledge of the genetic basis of human complex diseases compared with the pre GWAS approaches. Already Genome wide studies have identified over 300 genes associated with cardiovascular diseases in the last few years. It is needful to investigate other genetic variations using Genome wide studies to discover additional disease-associated genes to explain the heritability of hypertension among the peoples of Calabar and Uyo, Nigeria. Contributions to knowledge: In this study, the ethnic populations in Calabar and Uyo were screened for 3 polymorphisms associated with genes of the of the renin- angiotensin aldosterone system RAAS. Only one of the polymorphism, the Insertion/Deletion polymorphism of the angiotensin converting enzyme ACE gene was significantly associated with hypertension. Thus the ACE gene polymorphism is a molecular marker for hypertension in the study population. Angiotensinogen levels in plasma samples of participants was also measured the mean O.D. values for angiotensinogen was significantly higher in the patients than in the control when the O.D. vales were compared between patients and controls using student T test. The population was also screened for another polymorphism associated with hypertension, the atrial natriuretic peptide gene which plays an important role in a system that is in contrast to the RAAS. The mutant allele was not observed in the study population. Enviromental factors that are predisposing factors to hypertension were studied. Except for age and gender, all other factors were not significant predisposing factors for hypertension in the study population when regression analysis was carried out. This research will form baseline information for subsequent molecular studies in this population. CONCLUSION In this study, the frequency of the M235T variant of the AGT gene is high particularly the AGT M235T homozygous genotype. The M235T mutation, the angiotensin II type 1 receptor gene and atrial natriuretic peptide gene are not independent risk factors for hypertension in the sample population. However the I/D polymorphism of the ACE gene was associated with an increased risk for hypertension in the sample population collected from Calabar and the Uyo. The C664G variant of the atrial natriuretic peptide gene was not observed in this population. Linkage disequilibrium was observed among the patients, this is inconclusive due to the heterogeneity of the study population. Blood pressure is a complex phenotype with many physiologic pathways and compensatory system such that any gene or polymorphism within a gene will only explain a small part of the 98 variability when studied in isolation from other genes and polymorphism acting in concert to control blood pressure. Due to the fact that blood pressure is a physiologic series of checks and balances, it might be more meaningful to study the genetic effect of a group of genes acting on a particular physiologic pathway in a large more homogenous population to make definite conclusions. 99 REFERENCES Addo, J.A., Ama, A.G., Koram, K.A. 2006. The changing pattern of hypertension in Ghana, a study of four rural communities in the Ga district. Ethnicity and Disease.16: 894- 899. Addo, J., Smeeth, L. and Leon, D.A. 2007 Hypertension in sub Saharan Africa: a systematic review Hypertension 50: 1012 – 1018. Adedoyin, R.A., Mbada, C.E., Awofolu, O.O. and Oyebami, O.M. 2005 The influence of socio- economic status on casual blood pressure of the adult Nigerians. European Journal of Cardiovascular Prevention and Rehabilitation. 12.3: 271-273. Adedoyin, R.A., Mbadab, C.E., Balogun, M.O., Martins, T., Adebayo, R.A., Akintomide, A., Akinwusi, P.O. 2008. Prevalence and pattern of hypertension in a semi urban community in Nigeria. European Journal of Cardiovascular Prevention Rehabilitation. 15.6: 683-687. Adrogué, H.J. and Madias, N.E. 2007 Sodium and Potassium in the Pathogenesis of Hypertension. The New England Journal of Medicine. 356: 1966-1978 Agachan, B., Isbir, T., Yilmaz, H., Akoglu, E. 2003. Angiotensin converting enzyme I/D, angiotensinogen T174M – M235T and angiotensin II type 1 receptor gene polymorphism in Turkish hypertensives. Experimental and Molecular Medicine. 35:545-549. Akinkugbe, O.O. 1992. Tropical nephropathy: an overview. African Journal of Medicine and Medical Sciences 21.1: 3–7. Akinkugbe, O.O. 2000. Current epidemiology of hypertension in Nigeria. Archives of Ibadan Medicine 1.1: 3-5. + Aldred, K. L., Harris, P. J. and Eitle, E. 2000: Increased proximal tubule NHE-3 and H - ATPase activities in spontaneously hypertensive rats. Journal of Hypertension 18: 623–628. Aligbe, J.U., Akhiwu, W.O., Nwosu, S.O. 2002. Prospective study of coroner‟s autopsies in Benin City, Nigeria. Medicine, Science and the Law. 42.4: 318–324. Alvarez, R., Reguero, J.R., Batalla, A., Iglesias-Cuero, G., Cortina, A., Alvarez, V., Coto, E., 1998. Angiotensin coverting enzyme and angiotensin II receptor 1 polymorphism- association with early coronary disease Cardiovascular Research 40:375-379 Amakiri, C.N., Akang, E.E., Aghadiuno, P.U. and Odesanmi, W.O. 1997. A prospective study of coroner‟s autopsies in University College Hospital, Ibadan, Nigerian Medicine, Science and the Law. 37.1: 69–75. Angius, A., Petretto, E., Maestrale, G.B., Forabosco, P., Casu, G., Piras, D., Franciulli, M., Falchi, M., Mellis, P.M., Palermo, M. and Pirasty, M. 2002. A New Essential Hypertension Susceptibility Locus on Chromosome 2P 24-25 Detected by Genome wide Search. American Journal Human Genetic 71.4: 893-905. 100 Ashraf, S. S., Guenther, R. and Agris, P. F. 1999. Orientation of the tRNA anticodon in the ribosomal P-site: quantitative footprinting with U33-modified, anticodon stem and loop domains. RNA 5: 1191–1199. Atwood, L. D., Kammerer, C. M., Samollow, P. B., Hixson, J. E., Shade, R. E. and MacCluer, J. W. 1997. Linkage of essential hypertension to the angiotensinogen locus in Mexican Americans. Hypertension 30: 326–330. Awoyemi, A.O., Osagbemi, G.K. and Ogunleye, V.A. 2001. Medical examination findings among army recruits in Ilorin. West African Journal of Medicine. 20.3: 256- 258. Azizi, M., Guyene, T. T., Chatellier, G., Wargon, M. and Menard, J. 1997. Additive effects of losartan and enalapril on blood pressure and plasma active renin. Hypertension 29: 634–640. Badaruddoza, A.J.S., Bhanwer, R., Sawhney, N.K., Randhawa, K. Matharoo, K. and Barna, B. 2009 A Study of Angiotensin Converting Enzyme (ACE) Gene Polymorphism in Essential Hypertension among a Business Community in Punjab. International Journal of Human Genetics 9(3-4): 231-234. Basak, A.A., Sipahi, T., Ustundag, S., Ozgen, M., Sen, S. and Sener, S. 2008. Association of angiotensinogen T174M and M235T gene variants with development of hypertension in Turkish subjects of Trakya region. Biotechnology and Biotechnology EQ 984-989. Batzer, M.A. and Deininger, P.L. 2002 Alu repeats and human genomic diversity. Nature Reviews Genetics 3:370-379. Bell, A.C., Adair, L.S. and Popkin, B.M. 2002. Ethnic differences in the association between body mass index and hypertension. 155.4: 346-353. Bella, A.F., Baiyewu, O., Bamigboye, A., Adeyemi, J.D., Ikuesan, B.A. and Jegede, R.O. 1993 The pattern of medical illness in a community of elderly Nigerians. Central African Journal of Medicine. 39.6: 112–116. Benetos, A., Gautier, S., Ricard, S., Topouchian, J. and Cambien, F. 1996. Influence of angiotensin- converting enzyme and angiotensin II type 1 receptor gene polymorphism on aortic stiffness in normotensives and hypertensives patients. Circulation 94:698-703. Benjafield, A. V., Wang, W. Y. and Morris, B. J. 2004 No association of angiotensin- converting enzyme 2 gene (ACE2) polymorphisms with essential hypertension. American Journal of Hypertension 17: 624–628 Bereczky, S., Martensson, A., Pedro-Gil, J. and Farnert, A. 2005. Short report: rapid DNA extraction from archival blood spots on filter paper for genotyping of Plasmodium falciparum. American Journal of Tropical Medicine and Hygiene 72.3: 249-251. 101 Bhavani, B.A., Padma, T., Shastry, B.K.S. and Reddy, N.K.S. 2004 Gender specific association on insertion/deletion polymorphism of the human angiotensin converting enzyme gene with essential hypertension. International Journal of Human Genetics 4:207-13. Biron P, Mongeau J.G. and Bertrand D.1976. Familial aggregation of blood pressure in 558 adopted children. Canadian Medical Association Journal 115: 773-774. Bloem, L.J., Foroud, T.M, Ambrosus, W.T., Hanna, M.P., Tewsbury, D.A. and Pratt, J.H 1997. Association of the angiotensinogen gene to serum angiotensinogen in blacks and whites. Hypertension 29: 1078-1082. Bonnardeaux, A., Davies, E., Jeunemaitre, X., Fery. I., Charru, A., Clauser, E., Tiret, L., Cambien, F., Corvol, P. and Soubrier, F. 1994. Angiotensin II type 1 receptor gene polymorphism in human essential hypertension. Hypertension 24:63-69 Boss, O., Hagen, T. and Lowell, B. B. 2000. Uncoupling proteins 2 and 3: potential regulators of mitochondrial energy metabolism. Diabetes 49: 143–156. Brand, E., Chatelain, N. and Keavney, B. 1998. Evaluation of the angiotensinogen locus in human essential hypertension: a European study. Hypertension 31: 725–729. Brandin, L., Bergstrom, G., Manhem, K. and Gustafsson, H. 2003. Oestrogen modulates vascular adrenergic reactivity of the spontaneously hypertensive rat. Journal of Hypertension 21: 1695–1702. Burket, B.A 2006. Blood pressure survey in two communities in the Volta region of Ghana, West Africa. Ethnicity and Disease 16: 292 - 294. Caulfield, M., Lavender, P., Farral, M., Path, P., Munroe, P., Lawson, M., Turner, P., Lark, A .J .L. 1994 Linkage of the angiotensinogen gene to essential Hypertension. New England Journal Medicine 330: 1629-1633. Caulfield, M., Lavender, P., Newell-Price Farrall, M., Kamdar, S., Daniel, H., Lawson, M., Frectas, D., Fogarty, P., and Clark, A. J. 1995. Linkage of the angiotensinogen gene locus to human essential hypertension in African Caribbeans. Journal of Clinical Investigation 96.2: 687-692. Chiang, F.T., Hsu, K.L., Tseng, C.D., Hsiao W.H., Lo, H. M., Chern, T.H and Tseng, Y.M. 1997. Molecular variant M235T of the angiotensinogen gene is associated with essential hypertension in Taiwanese. Journal of Hypertension 15: 607-611. Chmaisse, H.N., Jammal, M., Fakhoury, H. and Fakhoury, R. 2009 A study on the association between Angiotensin 1 converting enzyme I/D dimorphism and type 2 diabetes. Saudi Journal of kidney disease and implantation. 20(6):1038-1046 Cooper R.S., Charles N.R. and Ward R. 1999. The Puzzle of Hypertension in African Americans, Scientific America. 36-43 Sciamdigital.com 102 Cooper, R., Forrester, T., Ogunbiyi, O., Muffinda, J., 1998. Angiotensinogen levels and obesity in four black populations. ICSHIB Investigators. Journal of Hypertension 16.5: 571-575. Cooper, R., Puras, A., Tracy, J. 1997a. evaluation of an electronic blood pressure device for epidemiological studies. Blood Pressure Monitoring 2: 35-40. Cooper, R., Rotimi, C., Ataman, S 1997b. The prevalence of hypertension in seven populations of West African origin. American Journal of Public Health. 87: 160-168. Cooper, R.S., Guo, X., Rotimi, C.N., Adeyemo, A. and Danilou, S.M. 2000. Heritability of Angiotensin converting enzyme and angiotensinogen: A comparison of US blacks and Nigerians. Hypertension 35: 1141- 1147. Cooper, R.S., Wolf- Maier, K., Luke, A. 2005. An International comparative study of blood pressure in populations of European versus African descent. BioMed Central Medicine 3: 2. Corvol, P., Persu, A., Gimenez-Roqueplo, A. and Jeunemaitre, X. 1999. Seven lessons from two candidate genes in human essential hypertension: angiotensinogen and epithelial sodium channel. Hypertension 33: 1324-1331 Corvol P. and Jeunemaitre X. 1997. Molecular Genetics of Human Hypertension: Role of Angiotensinogen 1. Endocrine Reviews 18.5: 662-677. Cui, J., Melista, E., Chazaro, I. 2005. Sequence variation of bradykinin receptors B1 and B2 and association with hypertension. Journal of Hypertension 23: 55–62 Danková, Z., Simáková, D., Luptáková O. and Blazícek, P. 2009 Association of ACE(I/D) polymorphism with metabolic syndrome and hypertension in two ethnic groups in Slovakia Anthropol Anz 67(3):305-316. Danser, A.H. and Schunket, H. 2000 Renin-angiotensin system gene polymorphism: potential mechanism for their association with Cardiovascular diseases. European Journal of Pharmacology 410:303-316 Das, M., Pal, S. and Ghosh, A. 2008 Angiotensin converting enzyme gene polymorphism Insertion/Deletion and hypertension in adult Asian Indians. A population based study from Calcutta, India. Human Biology 80(3):303-312. De Gasparo, M., Catt, K.J., Inagami, T., Wright, J.W. and Unger, T.H 2000. International union of pharmacology XXIII. The angiotensin II receptors. Pharmacology 52:415- 472 de Lange, M., Spector, T. D. and Andrew, T. 2004. Genome-wide scan for blood pressure suggests linkage to chromosome 11, and replication of loci on 16, 17, and 22. Hypertension 44: 872–877. Dellaporta, S.L, Wood, J. and Hicks, J.B. 1983. A plant DNA minipreparation, version11. Plant Molecular Biology. Reporter 1:19-21 103 DeStefano, A. L., Gavras, H., Heard-Costa, N. 2001. Maternal component in the familial aggregation of hypertension. Clinical. Genetics 60: 13–21. Donoghue, M., Hsieh, F. and Baronas, E. 2000. A novel angiotensin-converting enzyme- related carboxypeptidase (ACE2) converts angiotensin I to angiotensin 1–9. Circulation Research. 87: E1–E9. Douglas, J.G., Bakris, G.L. and Epstein, M. 2003. Hypertension in African Americans working group. Management of high blood pressure in African Americans: consensus statement of the hypertension in African Americans working group of the International society on hypertension in blacks. Archives of Internal Medicine 163:525-541. Dries, D., Victor, R.G., Rame, E., Cooper, R.S., Wu, X., Zhu, X., Leonard, D., Ho, S., Post, W. and Drazner, M.H. 2005. Corin gene minor defined by 2 missense mutations is common in blacks and associated with high blood pressure and hypertension. Circulation 112: 2403- 2410. Dubey, R. K., Oparil, S., Imthurn, B. and Jackson, E. K. 2002. Sex hormones and hypertension. Cardiovascular Research 53: 688–708. Dzau, V.J., Ellison, K.E., Brody, T., Ingelfinger, J., Pratt, R.E. 1987. A comparative study of the distributions of renin and angiotensinogen messenger ribonucleic acids in rat and mouse tissues. Endocrinology 120: 2334–2338. Dzida, G., Sobstyl, J., Puzniak, A., Golon, P., Mosiewiez, J. and Hanzli, J. 2001 . Polymorphism of angiotensin-converting enzyme and angiotensin II type 1 gene in Essential hypertension in a polish population Medical Science Monitor 7(6): 1236- 1241 Edwards, R., Unwin, N. and Mungasi F. 2000 Hypertension prevalence and care in an urban and rural area of Tanzania. Journal of Hypertension 18: 145-152. Ekere, A.U., Yellowe, B.E. and Umune, S. 2005. Mortality patterns in the accident and emergency department of an urban hospital in Nigeria. Nigerian Journal of Clinical Practice. 8.1: 14–18. Eriksson, U., Danilczyk, U. and Penninger, J. M. 2002. Just the beginning: novel functions for angiotensin-converting enzymes. Current Biology 12: R745–R752. Esterbauer, H., Schneitler, C., Oberkofler, H. 2001. A common polymorphism in the promoter of UCP2 is associated with decreased risk of obesity in middle-aged humans. Nature Genetics 28: 178–183. Exner, D.V., Dries, D.L., Domanski, M.J. and Cohen, J.N. 2001 Lesser response to angiotensin converting enzyme inhibitor therapy in Blacks as compared with White patients with left ventricular dusfunction New England Journal of Medicine 344(18)1351-1357. Fabris, B., Bortoletto, M., Candido, R. 2005. Genetic polymorphisms of the renin- angiotensin-aldosterone system and renal insufficiency in essential hypertension. Journal of Hypertension 23: 309–316. 104 Falase, A.O, Cole, T.O., Osuntokun, B.O. 1974. Myocardial infarction in Nigerian. Tropical and Geographical Medicine 25.2: 147–150. Falase, A.O., Ayeni, O., Sekoni, G.A., Odia, O.J. 1983. Heart failure in Nigerian Hypertensives. African Journal of Medical Science 12:7–15 Falase, A.O, Cole, T.O. and Osuntokun, B.O. 1974. Myocardial infarction in Nigerian Tropical and Geographical Medicine 25.2: 147–150. Farrag, W., Eid, M., El-Shazly, S. and Abdullah, M. 2010 Angiotensin II type 1 receptor gene polymorphism and telomere shortening in essential hypertension. Molecular and Cellular Biochemistry. DOI:10.1186/1471-2261-10-23 Farrag, W., Eid, M., El-Shazy, S. and Abdullah, M. 2011. Angiotensin 11 type 1 receptor gene polymorphism and Telomere shortening in essential hypertension. Molecular and Cellular Biochemistry 351(1-2):13-18 Feinleib, M., Garrison, R.J., Fabsitz, R., Christian, J.C., Hrubec, Z., Borhani, N.O. 1977. The NHLBI twin study of cardiovascular disease risk factors: methodology and summary of results. American Journal of Epidemiology 106: 284-285. Felder, R. A., Sanada, H., Xu, J. 2002. G protein-coupled receptor kinase 4 gene variants in human essential hypertension. Proceedings of the National Academy of Sciences 99: 3872–3877. Fornage, M., Amos, C.I., Kardia, S., Sing, C.F., Turner, S.T. and Boerwinkle, E.1998. Variation in the region of the angiotensin-converting enzyme gene influences inter individual differences in blood pressure levels in young white males. Circulation. 97: 1773-9. Fornage, M., Turner, S.T., Sing, C.F. and Boerwinkle, E. 1995. Variation at the M236T locus of the angiotensinogen gene and essential hypertension, a population-based case control study from Rochester, Minnesota. Human Genetics 96: 295-300. Gaillard, I., Clauser, E. and Corvol, P. 1989. Structure of human angiotensinogen gene. DNA 8: 87–99. Gaillard-Sanchez, I., Mattei, M.G., Clauser, E., Corvol, P. 1990. Assignment by in situ hybridization of the angiotensinogen gene to chromosome band 1q42, the same region as human renin gene. Human Genetics 84: 341–343. Gainer, J.V., Stein, C.M., Neal, T., Vanghan, D.E. and Bran, N.J. 2001 Interactive effect of Ethnicity and ACE Insertion/Deletion polymorphism on vascular reactivity Hypertension 37:46-51. Gard, P.R. 2010. Implications of the angiotensin converting enzyme gene Insertion/Deletion polymorphism in health and disease: a snapshot review International Journal of Epidemiology and Genetics 1(2):145-157 Geller, D. S., Farhi, A. and Pinkerton, N. 2000. Activating mineralocorticoid receptor mutation in hypertension exacerbated by pregnancy. Science 289: 119–123. 105 Genain, C., Bouhnik, J., Tewksbury, D., Corvol, P., Me´nard, J. 1984. Characterization of plasma and cerebrospinal fluid human angiotensinogen and des-angiotensin I angiotensinogen by direct radioimmunoassay. Journal of Clinical Endocrinology and Metabolism 59: 478– 484. Giles,T., Aranda, J.M., Suh, D.C. and Frech-Tamas, F. 2007. Ethnic/racial variations in blood pressure awareness, treatment and control. Journal of Clinical Hypertension 9.5: 345-54. Gong, M. and Huber, N. 2006. molecular genetics of human hypertension. Clinical Sciences 110: 315 -326. Gong, M., Zhang, H., Schulz, H. 2003. Genome-wide linkage reveals a locus for human essential (primary) hypertension on chromosome 12p. Human Molecular Genetics 12: 1273–1277. Grassi, G. 2001. Renin-angiotensin-sympathetic crosstalks in hypertension: reappraising the relevance of peripheral interactions. Journal of Hypertension 19: 1713–1716. Guo, X., Rotimi, C., Cooper, R., Luke, A., Elston, R.C., Ogunbiyi, O., and Ward, R. 1999. Evidence of a major gene effect for angiotensinogen among Nigerians. Annals of Human Genetics 63: 293-300. Gupta, S., Agrawal, B.K., Goel, R.K. and Sehajpal, P.K. 2009 Angiotensin-converting enzyme gene polymorphism in hypertensive rural population of Haryana, India Journal of Emergencies, Trauma and Shock 2 (3): 150-154. Hahntow, I.N., Mairuhu, G.,van Valkengoed, I.C.M., Koopmas, R.P. and Michel, M.C. 2010. Are functionally related polymorphisms of the renin-angiotensin-aldosterone system gene polymorphisms associated with hypertension. BioMed Central Cardiovascular Disorders 10:23 Hannila-Handelberg, T., Kontula, K. and Tikkanen, I. 2005. Common variants of the b and g subunits of the epithelial sodium channel and their relation to plasma renin and aldosterone levels in essential hypertension. BioMed Central Medical Genetics 6: 4. Hansson, J. H., Schild, L., Lu, Y. 1995. A de novo missense mutation of the b subunit of the epithelial sodium channel causes hypertension and Liddle syndrome, identifying a proline-rich segment critical for regulation of channel activity. Proceedings of the National Academy of Sciences. 92: 11495–11499. Harrap, S. B. 2003. Where are all the blood-pressure genes? Lancet 361: 2149–2151. Hashimoto, N. and Goldstein, B. J. 1992. Differential regulation of mRNAs encoding three protein-tyrosine phosphatases by insulin and activation of protein kinase C. Biochemical and Biophysical Research Communication 188: 1305–1311. Hata, A., Namikawa, C., Sasaki, M., Sato, K., Nakamura, T., Tamura, K., and La Louel, J. M. 1994. Angiotensinogen as a risk factor for essential hypertension in Japan. Journal of Clinical Investigation 93.3: 1285-1287. 106 Hegele, R.A., Brunt, J.H and Connely, P.W. 1994. A polymorphism of the angiotensinogen gene associated with variation in blood pressure in a genetic isolate. Circulation 90: 2207-2212. Hegele, R.A., Harris, S.B., Hanley, A.J., Sun, F., Conelly, P.W. and Zinman, B. 1997. Angiotensinogen gene variation associated with variation in blood pressure in aboriginal Canadians. Hypertension 29: 1073-1077. Hilgers, K.F., Langenfield M.R.W, Schlaich M, Veelken R., Schmieder, R.E. 1999 1166 A/C polymorphism of the angiotensin II type one receptor gene and the response to short infusion of angiotensin II. Circulation 100: 1394 - 1399. Hirawa, N., Uehara, Y., Yamakado, M. 2002. Lipocalin-type prostaglandin D synthase in essential hypertension. Hypertension 39: 449–454. Hsieh, M.C., Lin, S.R., Hsieh, T.J., Hsu, C.H., Chen, H.C., Shin, S.J. and Tsai, J.H. 2000. Increased frequency of angiotensin converting enzyme DD genotype in patients with type II diabetes in Taiwan. Nephrology Dialysis Transplantation 15: 1008-1013. Hsueh, W.C., Mitchell, B.D., Schneider, J.L., Wagner, M.J., Bell, C.J., Nanthakumar, E. 2000. QTL influencing blood pressure maps to the region of PPH1 on chromosome 2q31-34 in Old Order Amish. Circulation 101: 2810-2816. Hu, C., Wu, C., Lee, J., Hsieh, C., Chieng, C., Chang, S. and Chang C. 2007 Association between polymorphism of ACD, B2AR, ANP and ENOS and cardiovascular disease, a community based study in the Matsu area. Clinical Chemistry and Laboratory Medicine 45(1):20-25. Humayun, A., Shah, A.S. and Sultana, R. 2009 Relation of hypertension with body mass index and age in male and female population of Peshuwar, Pakistan. Journal of Ayub Medical College Abottabad 21(3):63-65. Hunt, S.C., Ellison, R.C, Atwood, L.D, Pankow, J.S., Province, M.A. and Leppert, M.F. 2002. Genome scans for blood pressure and hypertension: the National Heart, Lung, and Blood Institute Family Heart Study. Hypertension 40: 1-6. Ibrahim, M.M. 1996. The Egyptian national hypertension project (NHP) preliminary results. Journal of Human Hypertension 10.suppl.1: s39-41. Ike, S.O. and Onwubere, B.J. 2003. The relationship between diastolic dysfunction and level of blood pressure in Blacks. Ethnicity and Disease 13.4: 463–469. Iliadou, A., Lichtenstein, P., Morgenstern, R., 2002. Repeated blood pressure measurements in a sample of Swedish twins: heritabilities and associations with polymorphisms in the renin-angiotensin-aldosterone system. Journal of Hypertension 20, 1543-1550. Inoue, I., Nakajima, T. and Williams, C. S. 1997. A nucleotide substitution in the promoter of human angiotensinogen is associated with essential hypertension and affects basal transcription in vitro. Journal of Clinical Investigation 99: 1786–1797. 107 Isa, M.N., Boyd, E., Morrison, N., Harrap, E., Clauser, E. and Connor, J.M. 1990. Assignment of the human angiotensinogen gene to chromosome 1q42–q43 by non isotopic in situ hybridization. Genomics 8: 598–600. Isami, S., Kishikawa, H. and Araki, E. 1996. Bradykinin enhances GLUT4 translocation through the increase of insulin receptor tyrosine kinase in primary adipocytes: evidence that bradykinin stimulates the insulin signalling pathway. Diabetologia 39: 412–420. Ishigami, T., Umemura, S. and Tamura, K. 1997. Essential hypertension and 5´ upstream core promoter region of human angiotensinogen gene. Hypertension 30: 1325–1330. Ismail, M., Akhtar, N., Nasir, M., Firasat, S., Ayub, Q. and Khaliq, S. 2004 Association between the angiotensin converting enzyme gene Insertion/Deletion polymorphism and essential hypertension in Young Pakistani patients. Journal of Biochemistry and Molecular Biology 37:552-555. Iwai, N., Shimoike, H., Ohmichi, N. and Kinoshita, M. 1995. Angiotensinogen gene and blood pressure in the Japanese populations. Hypertension 25: 688-693. Iwamoto, T., Kita, S., Zhang, J., Blaustein, M.P., Yoshida, S. and Katsuragi, T. 2004. Salt sensitive hypertension is triggered by Ca2+ exchanger type-1 in vascular smooth muscle. Nature Medicine 10: 1993- 1199. Iyalomhe, G.B.S., Omogbai, E.K.L., Ozuola, R.I., Dada, F.L. and Iyalomhe, O.O.B. 2008. Electrolyte profiles in Nigerian patients with essential hypertension. African Journal of Biotechnology 7.10: 1404 – 1408. Jacobs, K. B., Gray-McGuire, C., Cartier, K. C. and Elston, R. C. 2003. Genome-wide linkage scan for genes affecting longitudinal trends in systolic blood pressure. BioMedCentral Genetics. 4: Suppl. 1, S82. Jafar, T.H., Chaturvedi, N. and Papps, G. 2006 Prevalence of Overweight and Obesity and their association with hypertension and diabetes mellitus in an Indo-Asian population. Canadian Medical Association Journal 175:1071-1077 Jain, S., Tang, X., Narayanan, C.S., Agarwal, Y., Ott, J. and Kumar, A. 2002. Angiotesinogen gene polymorphism at -217 affects basal promoter activity and is associated with hypertension in African- Americans. Journal of Biological Chemistry 277: 36889-36896. James, K., Weitzel, L. R., Engelman, C. D., Zerbe, G. and Norris, J. M. 2003. Genome scan linkage results for longitudinal systolic blood pressure phenotypes in subjects from the Framingham Heart Study. BioMedCentral Genetics. 4: Suppl. 1, S83. Jeunemaitre, X., Lifton, R. P., Hunt, S. C., Williams, R. R. and Lalouel, J. M. 1992a. Absence of linkage between the angiotensin converting enzyme locus and human essential hypertension. Nature Genetics 1: 72–75. Jeunemaitre, X., Rigat, B., Charru, A., Houot, A. M., Soubrier, F. and Corvol, P. 1992b. Sib pair linkage analysis of renin gene haplotypes in human essential hypertension. Human Genetics 88: 301–306. 108 Jeunemaitre. X., Soubrier, F., Kotelevtsev, Y.V., Lifton, R.P., Williams, C.S., Charru, A., Hunt, S.C., Hopkins, P.N., Williams, R.R., Lalouel, J-M., Corvol, P. 1992c. Molecular basis of human hypertension. Role of angiotensinogen. Cell 71: 169–180. Ji, Q., Ikegami, H., Fujisawa, T. 2004. A common polymorphism of uncoupling protein 2 gene is associated with hypertension. Journal of Hypertension 22: 97–102. Ji, L., Zhang, L., Shen, P., Wang, P., Zhang, Y., Xing, W., and Xu, J. 2010 Association of angiotensinogen converting enzyme gene I/D polymorphism with essential hypertension in Han Chinese population a meta analysis. Journal of Hypertension 28(3):419-428. Jiang, Z., Zhao, W., Yu, F. and Xu, G. 2001. Association of angiotensin II type 1 receptor gene polymorphism with essential hypertension. Chinese Medical Journal 114, 1249- 1251. John, U., Meyer, C., Hanke, M., Völzke, H. and Schumann, A., 2006. Smoking Status, Obesity and hypertension in a general population sample: a cross sectional study. Oxford Journal of Medicine 99.6: 407-415. Johnson, J.A. 2008 New drugs and technologies: Ethnic differences in cardiovascular drug response Circulation 118:1383-1393. Jose, P. A., Eisner, G. M. and Felder, R. A. 1998. Regulation of D1 receptor function in spontaneous hypertension. Advanced Pharmacology 42: 525–528. Jose, P. A., Eisner, G. M. and Felder, R. A. 2003 Dopamine and the kidney: a role in hypertension? Current Opinion in Nephrology and Hypertension 12: 189–194. Jurka, J. 2004 Evolutionary impact of human Alu repetitive elements. Current Opinion in Genetics and Development 14:603-8. Kadiri, S. 2000. Management of Hypertension with special emphasis on Nigeria In Archives of Ibadan medicine Olaopa E.O. Edi Hypertension Edition Archives of Ibadan medicine 1.1: 19-21. Kageyama, R., Ohkubo, H. and Nakanishi, S. 1984. Primary structure of human pre- angiotensinogen deduced from the cloned cDNA sequence. Biochemistry 23: 3603– 3609 Kamide, K., Kokubo, Y. and Yang, J. 2005. Hypertension susceptibility genes on chromosome 2p24-p25 in a general Japanese population. Journal of Hypertension 23: 955–960. Kannel, W. B. and Higgins, M. 1990. Smoking and hypertension as predictors of cardiovascular risk in population studies. Journal of Hypertension Suppl. 8: S3–S8. Kaplan, N.M. 2006. Primary hypertension: pathogenesis. Kaplan's clinical hypertension. Kaplan N.E. Eds 9th Edition. Philadelphia: lippincott Williams & Wilkins. 50- 121. 109 Kardia, S. L., Rozek, L. S., Krushkal, J. 2003. Genome-wide linkage analyses for hypertension genes in two ethnically and geographically diverse populations. American Journal of Hypertension 16: 154–157. Kario K., Kanai, N., Saito, N., Nago, N., Matsuo, T. and Shimada, K. 1996. Ischemic Stroke and the gene for angiotensin- converting enzyme in Japanese hypertension Circulation 93:1630-1633 Kato, N., Ikeda, N., Nabika, T., Morita, H., Sugiyama, T. and Gotoda, T. 2002 Evaluation of the atrial natriuretic pepetide gene in stroke Atherosclerosis. 163:279-286. Kato, N., Sugiyami, T., Morita, H., Nabikas, T., Kurihara, H., Yamori, Y. and Yazaki, Y. 2000. Genetic analysis of the atrial natriuretic peptide gene in essential hypertension. Clinical Science 98:251-258. Katsurada, A., Hagiwara, H., Miyashita, K., Satou, R., Miyata, K. and Kobori, H. 2007 Novel sandwich ELISA for human angiotensinogen. American Journal of Renal Physiology 293.3: 956-960. Katsuya, T., Horiuchi, M., Chen, Y.D.I., Koike, G., Pratt, R.E. and Dzau, V. 1995. Relations between deletion polymorphism of the angiotensin-converting enzyme gene and insulin resistance, glucose intolerance, hyperinsulinemia, and dyslipidemia. Arteriosclerosis Thromobosis and Vascular Biology 15: 779-782. Kaufman, J.S. and Barkey, N 1993. Hypertension in Africa: an overview of prevalence rates and causal risk factors. Ethnicity and Disease suppl S83-101. Kaufman, J.S., Rotimi, C.N., Brieger, W.R. 1996. The mortality risk associated with hypertension: preliminary results of a prospective study in rural Nigeria. Journal of Human Hypertension 10.7: 461–464. Kaufman, J.S., Owoaje, E.E., Rotimi, C.N., 1999. Blood pressure change in Africa: case study from Nigeria. Human Biology 71: 641-657. Ketterer, B., Harris, J. M. and Talaska, G. 1992. The human glutathione S-transferase supergene family, its polymorphism, and its effects on susceptibility to lung cancer. Environmental Health Perspectives 98: 87–94. Khogali, S. S., Mayosi, B. M., Beattie, J. M., McKenna, W. J., Watkins, H. and Poulton, J. 2001 A common mitochondrial DNA variant associated with susceptibility to dilated cardiomyopathy in two different populations. Lancet 357: 1265–1267. Kikuya, M., Sugimoto, K., Katsuya, T., Suzuki, M., Sato, T., Imai, Y. and Matsubara M. 1166 2003 A/C gene polymorphism of the angiotensin II type 1 receptor (AT1) and ambulatory blood pressure: the Ohasama study. Hypertension research 26:141-145. Knowles, A. F., Guillory, R. J. and Racker, E. 1971. Partial resolution of the enzymes catalyzing oxidative phosphorylation. XXIV. A factor required for the binding of mitochondrial adenosine triphosphatase to the inner mitochondrial membrane. Journal of Biological Chemistry 246: 2672–2679. 110 Koivukoski, L., Fisher, S. A. and Kanninen, T. 2004. Meta-analysis of genome-wide scans for hypertension and blood pressure in Caucasians shows evidence of susceptibility regions on chromosomes 2 and 3. Human Molecular Genetics 13: 2325–2332. Kunz, R., Kreutz, R., Biege, I., Dister, A. and Sharma, A.M. 1997 Association between angiotensinogen 235T variant and essential hypertension in Whites: a systematic review and methodological appraisals. Hypertension 30:1331-1337. Laivuori, H., Lahermo, P. and Ollikainen, V. 2003 Susceptibility loci for preeclampsia on chromosomes 2p25 and 9p13 in Finnish families. American Journal of Human Genetics 72: 168–177. Lalouel, J. M. and Rohrwasser, A. 2001 Development of genetic hypotheses in essential hypertension. Journal Human Genetics 46: 299–306. Lapierre, A.C., Arce, M.E., Lopez,, J.R. and Ciuffo, G.M. 2006. Angiotensin II type1 receptor gene polymorphism and essential hypertension in San Luis. Biocell 30(3):447-455. Lee, K.B and Kim, U.K. 2003. Angiotensinogen and angiotensin II type 1 receptor gene polymorphism in patients with autosomal dominant polycystic kidney disease: effect on hypertension and ESRD. Yonse Medical Journal 44(4):641-647 Liberatos, P., Link, B.G. and Kelsey, J.L. 1988 The measurement of social class in epidemiology. Epidemiological Reviews 10:87-121 Lifton, R. P., Dluhy, R. G., Powers, M. 1992. A chimaeric 11b-hydroxylase/aldosterone synthase gene causes glucocorticoid-remediable aldosteronism and human hypertension. Nature 35: 262–265. Lifton, R.P., Gharavi, A.G. and Geller, D.S. 2001. Molecular mechanisms of human hypertension. Cell 104: 545-56. Lin, C. Y., Strom, A., Vega, V. B. 2004. Discovery of estrogen receptor a target genes and response elements in breast tumor cells. Genome Biology 5: R66. Liu, Y., Zhouma, C., Shan, G., Cui, C., Hou, S., Qin, W., Gesang, L., Zhou,W. and Qiu, C. 2002 A1166C polymorphism of the angiotensin II type 1 receptor gene and essential hypertension in Han, Tibetan, and Yi populations. Hypertension Research 25:515- 521 Liu, W., Zhao, W. and Chase, G. A. 2004. Genome scan meta-analysis for hypertension. American Journal of Hypertension 17: 1100–1106. Lloyd-Jones, D.M., Evans, J.C and Levy, D. 2005. Hypertension in Adults across the age spectrum. Journal of the American Medical Association 294: 466-472. Longini, I.M. Jr, Higgins, M.W., Hinton, P.C., Moll, P.P. and Keller, J.B. 1984. Environmental and genetic sources of familial aggregation of blood pressure in Tecumseh, Michigan. American Journal of Epidemiology 120: 131-144. 111 Luft FC. 2000. Molecular genetics of human hypertension. Current Opinion in Nephrology and Hypertension 9: 259-66. Lynch, A. I., Boerwinkle, E., Davis, B. R., Ford, C. E., Eckfeldt, J. H., Leiendecker-Foster, C., and Arnett, D. K. 2008. Pharmacogenetic association of the NPPA T2238C genetic variant with cardiovascular disease outcomes in patients with hypertension. The journal of the American Medical Association, 299(3): 296 McCrindle, B.W. 2010 Assesment and management of hypertension in children and adolescents. Nature Reviews Cardiology 7:155-163. Mc Donagh, E.M., Whirl- Carrilo, Y., Garten, R.B., Altman, T and Klein, T.E. 2011 From pharmocogenomic knowledge acquisition to clinical application, the PharmGKB as a clinical pharmacogenomic biomarker resource. Biomarkers in Medicine 5(6):795-806. Markovic, D., Tang, X., Guruju, M., Levenstien, M.A., Hoh, J., Kumar, A. and Ott, J. 2005. Association of angiotensinogen gene polymorphisms with essential hypertension in African –Americans and Caucasians. Human Heredity 60: 89-96. Martinez, E., Pura, S.A., Escribano, J., Sanchis, C., Carrion, L., Masso, J. and Fernandez, J.A. 2002. Threonine at position 174 and 235 of the angiotensinogen polypeptide chain are related to familial history of hypertension in a Spanish – Meditteranean population. British Journal of Biomedical Sciences 59: 95 -100. Mayan, H., Munter, G. and Shaharabany, M. 2004. Hypercalciuria in familial hyperkalemia and hypertension accompanies hyperkalemia and precedes hypertension: description of a large family with the Q565E WNK4 mutation. Journal of Clinical Endocrinology and Metabolism 89: 4025–4030. Melander, O., Orho, M. and Fagerudd, J. 1998. Mutations and variants of the epithelial sodium channel gene in Liddle's syndrome and primary hypertension. Hypertension 31: 1118–1124. Mellen, P.B. and Herrington D.M. 2005 Pharmacogenomics of blood pressure response to antihypertensive treatment Journal of Hypertension 23(7):1311-1325. Mendelsohn, M. E. and Rosano, G. M. C. 2003 Hormonal regulation of normal vascular tone in males. Circulation Research 93: 1142–1145. 112 Mercer, E. A., Korhonen, L., Skoglosa, Y., Olsson, P. A., Kukkonen, J. P. and Lindholm, D. 2000. NAIP interacts with hippocalcin and protects neurons against calcium- induced cell death through caspase-3-dependent and -independent pathways. EMBO Journal 19: 3597–3607. Merkus, D., Duncker, D. J. and Chilian, W. M. 2002. Metabolic regulation of coronary vascular tone: role of endothelin-1. American Journal of Physiology – Heart and Circulatory. Physiology 283: H1915–H1921. Miwa, Y., Takiuchi, S., Kamide, K. 2004. Identification of gene polymorphism in lipocalin- type prostaglandin D synthase and its association with carotid atherosclerosis in Japanese hypertensive patients. Biochemical and Biophysical Research Communications 322: 428–433. Morisawa, T., Kishimoto, Y., Kitano, M., Kawasaki, H. and Hascgawa, J. 2001. Influence of angiotensin II type 1 receptor polymorphism on patients with hypercholesterolemia. Clinca Chimica Acta 304:91-97 Morris, R.C. Jr, Sebastian, A., Forman, A., Tanaka, M. and Schmidlin, O. 1999. Normotensive salt sensitivity: effects of race and dietary potassium. Hypertension 33:18-23. Morrise, T., Takeguchi, Y. and Takeda R. 1994 Angiotensin converting enzyme polymorphism and essential hypertension. Lancet 343:125 Morshed, M., Khan, H. and Akhteruzzaman, S. 2002 Association between angiotensin I – converting Enzyme gene polymorphism and hypertension in selected individuals of the Bangladeshi population. Journal of Biochemistry and Molecular Biology. 35:251– 254. Mufunda, J., Mebrahtu, G., Usman, A., Nyaran, P., Kosia, A., Ghebrat, Y., Ogbamanam A., Masjuan, M. and Gebremichael, A. 2006. The Prevalence of Hypertension and its relationship with obesity: results from a national blood pressure survey in Eritea. Journal of Human Hypertension 20: 59-65. Mune, T., Rogerson, F. M., Nikkila, H., Agarwal, A. K. and White, P. C. 1995. Human hypertension caused by mutations in the kidney isozyme of 11b-hydroxysteroid dehydrogenase. Nature Genetics 10: 394–399. Naber, C. K. and Siffert, W. 2004. Genetics of human arterial hypertension. Minerva Medica 95: 347–356. Newton-Cheh, C., Johnson, T., Gateva, V., Tobin, M.D., Bochud, M., Coin, L., Najjar, S.S., Zhao, J.H., Heath, S.C., Eyheramendy, S., Caulfield, M., Munroe, B.M. 2009 Genome-wide association study identifies eight loci associated with blood pressure. Nature Genetics 41:666-676. Nishiuma, S., Kario, K., Kayaba, K., Nagio, N., Shimada, K., Matsuo, T. and Matsuo, M.1995 Effect of the angiotensinogen gene Met235→Thr variants on blood pressure 113 and other cardiovascular risk factors in two Japanese population. Journal of hypertension 13: 717-722. Niu, T., Xu, X. and Rogus, J. 1998. Angiotensinogen gene and hypertension in Chinese. Journal of Clinical Investigation 101: 188–194 Niu, T, Yang, J., Wang, B., Chen, W., Wang, Z. and Laird, N. 1999. Angiotensinogen gene polymorphisms M235T/T174M: no excess transmission to hypertensive Chinese. Hypertension. 33: 698-702. O‟Donnell, C.J., Lindpaintner, K., Larson, M.G., Rao, V.S. and Ordovas, J.M. 1998. Evidence for association and genetic linkage of the angiotensin converting enzyme locus with hypertension and blood pressure in men but not women in the Framingham Heart Study. Circulation. 97: 1766-72. Ogah, O.S 2006 Hypertension in Sub –Saharan African populations. The burden of hypertension in Nigeria. Ethnicity and Disease 16: 765. Ogunlesi, A.O. 2000. Aetiopathogenesis of hypertension; Molecular Biology and Hypertension an overview in Archives of Ibadan medicine Olaopa E.O Edi Archives of Ibadan Medicine 1.1: 5-7. Ogunniyi, A., Baiyewu, O. and Gureje, O. 2001. Morbidity pattern in a sample of elderly Nigerians resident in Idikan community, Ibadan. West African Journal of Medicine 20.4: 227–231. Okojie, O.H., Isah, E.C. and Okoro, E. 2000. Assessment of health of senior executives in a developing country. Public Health. 114.4: 273–275. Ono, K., Mannami, T., Baba, S., Yasui, N., Ogihar, T., Iwa, N. 2003 Lack of association between angiotensin II type I receptor gene polymorphism and hypertension in Japanese Hypertension Research 26:131-134 Opadijo, O.G., Omotoso, A.B.O. and Akande, A.A. 2003. Relation of electrocardiographic left ventricular hypertrophy to blood pressure, body mass index, serum lipids and blood sugar levels in adult Nigerians. African Journal of Medicine and Medical Sciences 32: 395–399. Oparil, S., Zaman, M.A., and Calhoun, D.A. 2003. Pathogenesis of hypertension. Review. Annals of Internal Medicine 139: 761-776. + + Orlowski, J. and Grinstein, S. 1997. Na /H exchangers of mammalian cells. Journal of Biological Chemistry 272: 22373–22376. Osanai, T., Sasaki, S. and Kamada, T. 2003. Circulating coupling factor 6 in human hypertension: role of reactive oxygen species. Journal of Hypertension 21: 2323– 2328. Osanai, T., Tanaka, M. and Kamada, T. 2001. Mitochondrial coupling factor 6 as a potent endogenous vasoconstrictor. Journal of Clinical Investigation 108: 1023–1030. 114 Osuntokun, B.O., Bademosi, O., Akinkugbe, O.O., Oyediran, A.B. and Carlisle, R. 1979 Incidence of stroke in an African city: results from the stroke registry at Ibadan, Nigeria, 1973–1975. Stroke. 10.2: 205–207. Pereira, A.C., Mota, G.F., Cunha, R.S., Herbenhorff, F.L., Mill, J.K. and Krieger, J.E 2003 Angiotensinogen 235T allele “dosage” is associated with blood pressure phenotypes. Hypertension 41: 25-30. Procopciuc, L., Jebeleanu, G., Surcel, I. and Puscus, M 2002. Angiotensinogen gene M235T variant and pre-eclampsia in Romanian pregnant women. Journal of Cellular Molecular and Medicine 6.3: 283-288. Rae, J. M., Johnson, M. D., Scheys, J. O., Cordero, K. E., Larios, J. M. and Lippman, M. E. 2005. GREB1 is a critical regulator of hormone dependent breast cancer growth. Breast Cancer Research Treatment 92: 141–149. Ramachandran, V., Ismail, P., Stanslas, J., Shamsudin, N., Moin, S. and Jas, R.M. 2008 Association of insertion/deletion polymorphism of angiotensin-converting enzyme gene with essential hypertension and type 2 diabetes mellitus in Malaysian subjects. Journal of Renin-Angiotensin-Aldosterone System 9(4):208-214 Ranade, K., Hinds, D., Hsiung, C. A. 2003. A genome scan for hypertension susceptibility loci in populations of Chinese and Japanese origins. American Journal of Hypertension 16: 158–162. Rice, T., Cooper, R.S., Wu, X., Bouchard, C., Rankinen, T., Rao, D.C., Jaquish, C.E., Fabsitz, R.R., Province, M.A. 2006 Meta-analysis of genome-wide scans for blood pressure in African American and Nigerian samples. American Journal of Hypertension 19:270-274. Rigat, B., Hubert, C., Alhenc-Gelas, F., Cambien, F., Corvol, P. and Soubrier, F. 1990 An insertion/deletion polymorphism in the angiotensin I-converting enzyme gene accounting for half the variance of serum enzyme levels. Journal of Clinical Investigation 86(4):1343-1346. Rigat, B., Hubert, C. and Corvol, P. 1992 PCR detection of insertion/deletion polymorphism of the human angiotensin converting enzyme gene (DCP1) (dipeptidyl carboxypeptidase 1) Nucleic Acid Research 20:1433 Rodriguez-Cruz, E., Ettinger, L.M. and Gesser, I.H. 2010 "Hypertension" eMedicine Pediatrics: Cardiac Disease and Critical Care. Rotimi, C., Cooper, R., Ogunbiyi, O., Morrison , L., Ladipo, M., Tewksbury, D. and Ward, R. 1997 Hypertension, serum angiotensinogen, and molecular variants of the angiotensinogen gene among Nigerians. Circulation 95.10: 2348- 2350. Rotimi, C., Morrison, L., Cooper, R., Oyejide, C., Effiong, E., Ladipo, M., Osotemihen,B. and Ward, R. 1994. Angiotensinogen gene in human hypertension. Lack of an association of the T235 allele among the African –Americans. Hypertension 24: 591- 594. 115 Rotimi, C.N., Peras, A., Cooper, R., Mc Farlene-Anderson, N., Forrester, T., Ogunbiyi, M. L., and Ward, R. 1996. Polymorphisms of the rennin angiotensin genes among Nigerians, Jamaicans and African Americans. Hypertension 27: 558-563. Rubattu, S., Bigatti, G., Evangelista, A., Lanzani, C., Stanzione, R., Zagato, L., Manunta, P., Marchitti, S., Venturelli, V., Bianch,G., Volpe, M. and Paulo, S. 2006 Association of atrial natriuretic peptide and type A natriuretic peptide receptor gene polymorphism with left ventricular mass in human essential hypertension. Journal of the American College of Cardiology 48(3):499-505. Rubattu, S., Di Angelantonio, E., Stanzionc, R., Zanda, B., Evangelista, A., Pirisi, A., brunette, E and Volpe, M. 2004. Gene polymorphism of the renin- angiotensin- aldosterone system and the risk of ischemic stroke: a role of the A1166C/ AT1 gene variant. Journal of hypertension 22:2129-2134 Saadat, M. and Dadbine-Pour, A. 2005. Influence of polymorphism of glutathione S- transferase M1 on systolic blood pressure of normotensive individuals. Biochemical and Biophysical Research Communications 326: 449–454 Sagnella, G.A., Rothwell, M.J., Onipinla, A.K., Wicks, P.D., Cook, P.G. and Cappuccio, F. 1999. A population study of Ethnic variations in the ACE/I/D polymorphism: relationships with gender, hypertension and impaired glucose metabolism. Journal of Hypertension 17:657-664. Sakuma, T., Hirata, R.D. and Hiraia, M.H. 2004 Five polymorphisms in gene candidates for cardiovascular disease in Afro-Brazilian individuals. Journal of Clinical Laboratory Analysis 18:309-316. Salem, A.H. 2008 Distribution of Angiotensin converting enzyme Insertion/Deletion gene polymorphism among two Arab populations. Suez Canal University Medical Journal 11(1):125- 130. Sameer, A.S., Syeed, N., Tak, S.A., Bashir, S.M., Nissar, S. and Siddiqi, M. ACE I/D polymorphisms in hypertensive patients of Kasmiri population. 2010 Cardiology Research 1(1):1-7. Sanada, H., Jose, P. A., Hazen-Martin, D. 1999. Dopamine-1 receptor coupling defect in renal proximal tubule cells in hypertension. Hypertension 33: 1036–1042. Say, Y., Ling, K., Duraisamy, G., Isaac, S and Rosli, R. 2005. Angiotensinogen M235T gene variants and its association with essential hypertension and plasma rennin activity in Malaysian subjects. A case control study. BioMedCentral Cardiovascular Disorders 5: 7 Schmidt, S., Beige, S., Walla-Friedel, M., Michel, M.C., Sharma, A.M. and Ritz, E. 1997. A polymorphism in the gene for the angiotensin II type 1 receptor is not associated with hypertension. Journal of Hypertension 15, 1385-1388. Schultheis, P. J., Clarke, L. L. and Meneton, P. 1998a. Renal and intestinal absorptive + + defects in mice lacking the NHE3 Na /H exchanger. Nature Genetics 19: 282–285 116 Schultheis, P. J., Lorenz, J. N. and Meneton, P. 1998b. Phenotype resembling Gitelman's + - syndrome in mice lacking the apical Na -Cl cotransporter of the distal convoluted tubule. Journal of Biological Chemistry 273: 29150–29155. Schuster, H., Wienker, T. E. and Bahring, S. 1996. Severe autosomal dominant hypertension and brachydactyly in a unique Turkish kindred map to human chromosome 12. Nature Genetics 13: 98–100. Schwartz, F., Duka, A., Sun, F., Cui, J., Manolis, A. and Gavras, H. 2004. Mitochondrial genome mutations in hypertensive individuals. American Journal of Hypertension. 17: 629–635. Sethi, A. A., Nordestgaard, B. G., Agerholm-Larsen, B., Frandsen, E., Jensen, G. and Tybjarg-Hansen, A. 2001. Angiotensinogen and Elevated Blood Pressure in the General Population: The Copenhagen City Heart Study. Hypertension 37: 875-881. Sethi, A. A., Nordestgaard, B. G., Hanson, A. T. 2003. Angiotensinogen Gene Polymorphism and Risk of Hypertension and Ischemic heart Disease. A Meta- Analysis Journal of American Heart Association 23: 1269. Sharma, S. and Kortas, C. 2008. Hypertension. ℮medicine - Nephrology Shearman, A. M., Cupples, L. A. and Demissie, S. 2003. Association between estrogen receptor a gene variation and cardiovascular disease. Journal of American Medical Association 290: 2263–2270. Shearman, A. M., Cooper, J. A. and Kotwinski, P. J. 2005. Estrogen receptor a gene variation and the risk of stroke. Stroke 36: 2281–2282. Shimkets, R. A., Warnock, D. G. and Bositis, C. M. 1994. Liddle's syndrome: heritable human hypertension caused by mutations in the b subunit of the epithelial sodium channel. Cell 79: 407–414. Simard, J., Durocher, F. and Mebarki, F. 1996. Molecular biology and genetics of the 3b- hydroxysteroid dehydrogenase/D5-D4 isomerase gene family. Journal of Endocrinology 150: S189–S207. Speirs, H. J., Katyk, K., Kumar, N. N., Benjafield, A. V., Wang, W. Y. and Morris, B. J. 2004 Association of G-protein-coupled receptor kinase 4 haplotypes, but not HSD3B1 or PTP1B polymorphisms, with essential hypertension. Journal of Hypertension 22: 931–936. Staessen, J.A., Kuznetsova, T., Wang, J.G., Emelianov, D., Vlietinck, R. and Fagard, R. 1999. M235T angiotensinogen gene polymorphism and cardiovascular renal risk. Journal of Hypertensions 17: 9-17. Stamler, R., Stamler, J., Reidlinger, W.F., Algera, G. and Robert, R.H. 1978. Weight and Blood Pressure Journal of the American Medical Association 240.15: 1607-1610. Stankovic, A., Žickovic, M., Glišić, S. and Alavantić, D. 2003 Angiotensin 11 type 1 receptor gene polymorphism and essential hypertension in Serbian population Clinca Chimica Acta 327(1-20):181-185 117 Stergiou, G. S. and Salgami, E. V. 2004. New European, American and International guidelines for hypertension management: agreement and disagreement. Expert Reviews in Cardiovascular Therapy 2: 359–368. Stevens, J., Juheri, Cai, J. and Jones, D.W. 2002. The effect of decision rules on the choice of a body mass index cut off for obesity: examples from African American and White women. American Journal of Clinical Nutrition 75.6: 986-992. Stevens, J., Truesdale, K.P., Katz, E.G. and Cai, J. 2008. Impact of body mass index on incident hypertension and diabetes in Chinese, Asians, American Whites and American Blacks American Journal of Epidemiology 167.11: 1365- 1374. Stewart, P. M., Krozowski, Z. S., Gupta, A. 1996. Hypertension in the syndrome of apparent mineralocorticoid excess due to mutation of the 11b-hydroxysteroid dehydrogenase type 2 gene. Lancet 347: 88–91. Sugiyama, T., Morita, H., Kato, N., Kurihara, H., Yamori, Y. and Yazaki, Y. 1999. Lack of sex-specific effects on the association between angiotensin-converting enzyme gene polymorphism and hypertension in Japanese. Hypertension Research 22: 55–59. Sun, F., Cui, J., Gavras, H. and Schwartz, F. 2003. A novel class of tests for the detection of mitochondrial DNA-mutation involvement in diseases. American Journal Human Genetics 72: 1515–1526 Slatkin, M 2008 Linkage disequilibrium-understanding the evolutionary past and mapping the medical future. Nature Reviews Genetics 9:477-485. Sun, B., Dronma, T. and Qin, W. J. 2004. Polymorphisms of renin-angiotensin system in essential hypertension in Chinese Tibetans. Biomedical and Environmental Sciences 17: 209–216. Sunder-Plassmann, G., Kittler, H. and Eberle, C. 2002. Angiotensin converting enzyme DD genotype is associated with hypertensive crisis. Critical Care Medicine 30: 2236– 2241. Takami, S., Katsuya, T., Rakugi, H., Noriyuki, S. Nakata, Y., Kamitani, A., Higaki, J. and Ogihara, T. 1998. Angiotensin 11 type 1 receptor gene polymorphism is associated with increase of left ventricular mass but not with hypertension. American Journal of Hypertension 2:316-321 Tamura, H., Schild, L., Enomoto, N., Matsui, N., Marumo, F. and Rossier, B. C. 1996. Liddle disease caused by a missense mutation of b subunit of the epithelial sodium channel gene. Journal of Clinical Investigation 97: 1780–1784. Tedesco, M.A., Di Salvo, G., Caputo, S., Natale, F., Ratti, G., Iarussi, D. and Iacono, A. 2001 Educational level and hypertension: how socioeconomic differences condition health care. Journal of Human Hypertension 15:727-731 The Seventh Report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure (JNC 7). 2003. Journal of American Medical Association 289.19: 2560–2572. 118 Tiret, L., Blanc, H., Ruidavets, J.B., Arveiler, D., Luc, G., Jeunemaitre, X., Plouin, P.F. and Cambien, F. 1998. Gene polymorphism of the rennin angiotensin system in relation to hypertension and parental history of myocardial infarction and stroke: the PEGASE study. Journal of Hypertension 16:37-44 Tobina, T., Kiyonaga, A., Akagi, Y., Mori, Y., Ishii, K. and Chiba, H. 2007 Angiotensin 1 converting enzyme gene polymorphism and exercise trainability in elderly women: an electro-cardiological approach Journal of Sports Science and Medicine 6:220-226. Touyz, R. M. and Schiffrin, E. L. 2004. Reactive oxygen species in vascular biology: implications in hypertension. Histochemistry and Cell Biology 122: 339–352. Tsezou, A., Karayannis, G., Giannatou, E., Papanikolaou, V. and Triposkiadis F. 2008 Association of renin-angiotensin system and natriuretic peptide receptor A gene polymorphism with hypertension in a Hellenic population. Journal of Renin- Angiotensin-Aldosterone System 9:202-207. Tuan, N.T., Adair, L.S., Suchindran, C.M and Popkin, B.M. 2009 The association between body mass index and hypertension is different between East and South East Asians. American Journal of Clinical Nutrition 89:1905-1912. Uddin, M., Yang, H., Shi, M. 2003. Elevation of oxidative stress in the aorta of genetically hypertensive mice. Mechanisms of Ageing and Development 124: 811–817 Ulasi, I.I., Ijoma, C.K., Onwubere, B. J.C., Arodiwe, Onodugo, O. and Okafor, C. 2011 High prevalence and low awareness of hypertension in a market population in Enugu, Nigeria. International Journal of hypertension. doi:10.4061/2011/869675. Urade, Y., Fujimoto, N. and Hayaishi, O. 1985. Purification and characterization of rat brYen, C. H. and Lau, Y. T. 2004 17b-Oestradiol enhances aortic endothelium function and smooth muscle contraction in male spontaneously hypertensive rats. Clinical Science 106: 541–546. van Geel P.P., Pinto, Y.M., Buikema, H. and van Gislt, G.H. 1998 Is the A1166C gene polymorphism of the Angiotensin II type I receptor involved in cardiovascular disease? European Heart Journal 19:G13-G17. Van Geel, P.P., Pinto, Y.M., Voors, A.A., Buikema, H., Oosterga, M., Crijns, H.J and van 1166 Gilst, W.H. 2000 Angiotensin II type 1 receptor A C gene polymorphism is associated with an increased response to angiotensin II in human arteries. Hypertension 35(3):717-721 Vargas, C.M., Imgram, D.D. and Gillum, R.F. 2000 Incidence of hypertension and education attainment: the NHANES 1 epidemiologic follow up study First national health and nutrition examination survey. American Journal of Epidemiology 152:272- 278 Vasku, A., Soucek, M., Tschoplova, S. and Stejskalova, A. 2002. An association of BMI with A(-6)G, M235T and T174M polymorphisms in angiotensinogen gene in essential hypertension. Journal of Human Hypertension 16: 427–430. 119 Von Wowern, F., Bengtsson, K. and Lindgren, C. M. 2003. A genome wide scan for early onset primary hypertension in Scandinavians. Human Molecular Genetics 12: 2077– 2081. Wang , Y., Maggie, C.Y., Wing, Y.S., Peter, C.Y., Ronald, C.W., Chun, C.C., Clive, S.C. and Chan, J.C.N. 2005 Prognostic effect of Insertion/Deletion polymorphism of the ACE gene on renal and cardiovascular clinical outcomes in Chinese patients with type 2 diabetes. Diabetes Care 28(2):348-354 Wang, C. C., Goalstone, M. L. and Draznin, B. 2004. Molecular mechanisms of insulin resistance that impact cardiovascular biology. Diabetes 53: 2735–2740. Wang, J.G. and Staessen, J.A. Genetic polymorphisms in the renin-angiotensin system: relevance for susceptibility to cardiovascular disease. Eur J Pharmacol 2000;410(2- 3):289-302. Wang, W.Y., Zee, R.Y. and Morris, B.J 1997. Association of angiotensin II type 1 receptor gene polymorphism with essential hypertension. Clinical Genetics 51:31-34 Ward, K., Hata, A., Jeunemaitre, X., Helin, C., Nelson, L. Corvol, P., Lifton, R.P. and Lalouel, J.M. 1993. Molecular variant of angiotensinogen associated with preeclampsia. Nature Genetics 4: 59-61. Wassmann, S., Wassmann, K. and Nickenig, G. 2004 Modulation of oxidant and antioxidant enzyme expression and function in vascular cells. Hypertension 44: 381– 386. Watson, Jr, B., Khan, M. A., Desmond, R. A. and Bergman, S. 2001. Mitochondrial DNA mutations in black Americans with hypertension-associated end-stage renal disease. American Journal of Kidney Disease 38: 529–536. Weinberger, M.H., Fineberg, N.S., Fineberg, E. and Weinberger, M. 2001 Salt sensitivity, pulse pressure and death in normal and hypertensive humans. Hypertension 37:429- 432. Wigginton, J.E., Cutler, D.J. and Abecasis, G.R. 2005. A note on exact test of Hardy – Weinberg equilibrium. American Journal of Human Genetics 76: 887-893. Wilk, J. B., Djousse, L., Arnett, D. K. 2004. Genome-wide linkage analyses for age at diagnosis of hypertension and early-onset hypertension in the HyperGEN study. American Journal of Hypertension 17: 839–844. Wilson, F.H., Disse-Nicode`me, S., Choate, K.A., Ishikawa, K., Nelson-Williams, C. and Desitter, I. 2001. Human hypertension caused by mutations in WNK kinases. Science 293: 1107-1112. Wilson, F. H., Hariri, A. and Farhi, A. 2004. A cluster of metabolic defects caused by mutation in a mitochondrial tRNA. Science 306: 1190–1194. Wright, J.T. Jr., Dunn, J.K, Cutler, J.A. Davis, B.R., Cushman, W.C. and Ford, C.E. 2005. Outcomes in hypertensive Blacks and non Black patients treated with chlorthalidone, amlodipine, lisinopril. Journal of American Medical Association 293(13):1595-1608. 120 Wu, X., Kan, D., Province, M., Quertermous, T., Rao, D.D., Chang, C., Mosley, T.H., Curb, D., Boerwinkle, E. and Cooper, R.S. 2006. An updated meta-analysis of genome scans for hypertension and blood pressure in the NHLBI family blood pressure program (FBPP). American Journal of Hypertension 19:122-127. Xu, Q., Wang, Y. H. and Tong, W. J. 2004. Interaction and relationship between angiotensin converting enzyme gene and environmental factors predisposing to essential hypertension in Mongolian population of China. Biomedical and Environmental Sciences17: 177–186 Xue, H., Wang, S., Wang, H., Sun, K., Song, X., Zhang, W., Fu, C., Han, Y. and Hui, R. 2008 Atrial natriuretic peptide gene polymorphism is associated with left ventricular hypertrophy in hypertension. Clinical Science 114:131-137. Yang, H., Shi, M. and VanRemmen, H. 2003. Reduction of pressor response to vasoconstrictor agents by over expression of catalane in mice. American Journal of Hypertension 16: 1–5. Yen, C. H. and Lau, Y. T. 2004. 17b-Oestradiol enhances aortic endothelium function and smooth muscle contraction in male spontaneously hypertensive rats. Clinical Science 106: 541–546. Zee, R.Y., Lou, Y.K., Griffiths, L.R. and Morrise, B.J. 1992 Association of a polymorphism of angiotensin-I converting enzyme gene with essential hypertension. Biochemical and Biophysical Research Communication184:9-15. Zhang, Q., Cui, T.X. and Li, .L 2006 Investigation of relationship between gene polymorphism of angiotensin11 type 1 receptor and plasma nitric oxide and endothelin in essential hypertension. Medical Informatics 19:628-630 Zhenni, J., Wensheng, Z., Feng, Y. and Geng, Xu. 2001. Association of angiotensin II type 1 receptor gene polymorphism with essential hypertension. Chinese Medical Journal. 114(12):1249-1251 Zhou, X. F., Cui, J. and DeStefano, A. L. 2005. Polymorphisms in the promoter region of catalase gene and essential hypertension. Disease Markers 21: 3–7. Zhu, D. L., Wang, H. Y. and Xiong, M. M. 2001. Linkage of hypertension to chromosome 2q14-q23 in Chinese families. Journal of Hypertension. 19: 55–61. Zhu, H., Sagnella, G. A. and Dong, Y. 2004. Molecular variants of the sodium/hydrogen exchanger type 3 gene and essential hypertension. Journal of Hypertension 22: 1269– 1275. Zhu, X., Fejerman, L., Luke, A., Adeyemo, A. and Cooper, R.S. 2005. Haplotype produce from rare variants in the promoter and coding regions of angiotensinogen contribute to variation in angiotensinogen levels. Human Molecular Genetics 14.5: 639-64 Zhu, X., Yan, D., Cooper, R.S., Luke, A., Morna, A., Yen-Dec, C.C., Weder, A. and Chakravarti A. 2003. Linkage Disequilibrium and Haplotype Diversity in the Genes of the Renin Angiotensin system; findings from the Family Blood Pressure Program Retrieved October 6, 2006 from http// www.genome.org 121 122 APPENDICES Extraction buffer: To prepare 1000mls 100mM Tris ÷ 1000 = 0.1M 8.5mM EDTA ÷ 1000 = 0.0085M 500mM Nacl2 ÷ 0.5M Tris = 0.1 x 121.14 x 1000 = 12.114g 1000 EDTA = 0.0085 x 372.24x 1000 = 3.164g 1000 Nacl = 0.5 x 58.44 x 1000 = 29.22g 1000 The salts were weighed and added to about 500mls of distilled water, stirred until complete dissolution of the salts. Distilled water was added to make up to 1000 and the pH adjusted to 8.0. The solution was autoclaved. 20% Sodium dodecyl sulphate – SDS: 100ML 20G OF SDS salt was weighed and added to 60/80ml of distilled water. The solution was stirred for a while until complete dissolution of the salt. Distilled water was then added to make up to 100mls. 5M KOAC – Potassium Acetate using the formular Xg = molarity x molecular weight x volume (ml) 1000 = 5 x 98. 15 x 10ml 1000 = 4.9075 Salt (4.90) was weighed and added to 5ml of sterile distilled water, stirred until the salt dissolved completely sterile distilled water was added to make up to 10mls. Tris – Ethylene diamine tetra-acetate buffer (T.E.) buffer. 123 10mM Tris ÷ 1000 = 0.1M Tris 1mM EDTA ÷ 1000 = 0.001M EDTA Tris Xg = 0.01 x 121.14 x1000 = 1.211 1000 2 5000ml = 0.605g of salt EDTA Xg = 0.001 x 372.24 X 1000 = 0. 372 1000 2 500ml = 0.186 To prepare 500mls of T.E.: 0. 605g of Tris molecular grade and 0.186g of EDTA were added to 300mls of distilled water, stirred until complete dissolution of the salts and distilled water was then added to make 500mls and the solution was autoclaved. Tris – EDTA buffer for filter paper extraction. 10mM ÷ 1000 = 0.1M Tris base Xg = 0.1 x 121.14 x 500mls 1000 = 0.6057g of Tris 0.1mM EDTA ÷ 1000 = 0.0001m Xg = 0.0001 x 372.24 50mls 1000 = 0.0186 of EDTA. Both salts were weighed and added to 350mls of sterile distilled water, stirred until complete dissolution of salts. Then sterile distilled water was added to make up500mls. Reconstitution of Primer R 31.4mol x 10 = 314ul This amount of T.E F 32.7nmol x 10 = 327ul was added to the lyophilised powder. For working dilution 90ul of T.E was added to 10ul of reconstituted lyophilized powder to make 100ul. Gel preparation. 124 50 x TAE was diluted to 1 X by diluting 20mls of 50x in 1000ml of distilled water. The ends of the plastic tray supplied with the electrophoresis apparatus was sealed with adhesive tape. 100ml of 1 TAE was measured and put into a bottle; 1.5g of agarose powder was added and heated in the microwave oven for 5 min to dissolve the agarose. o The bottle was then allowed to cool to about 60 C; 5µl of ethidium bromide was added. When checking for DNA quality, 0.8g of agarose and 0.8ul of ethidium bromide was used. The agarose was poured into the tray and allowed to set at room temperature (30 – 45 mins). Preparation of 10mM dNTP from 100mM stock (dNTP components comes as 100Mm stock concentration, the kit is made up of: dATP; dCTP; dGTP and dTTP. For a volume of 500µl C1V1 = C2V2 100mM× V1 = 10mM×500 V1 = 10×500 100 V1 = 50µl 50µl of dATP; dCTP; dGTP and dTTP were measured making a total of 200µl, 300µl of sterile water was added to make up 500µl. Preparation of ELISA buffers. 4 Liters of Phosphate Buffer Saline – Tween (PBS-Tween) X10 pH 7.4 Sodium Chloride 320g Potassium phosphate (monobasic) 8g Sodium phosphate (diabasic) 44g Potassium chloride 8g Tween 20mls All the salts were measured and dissolved in distilled water, put on a magnetic stirrer for proper dissolution of the salts, the pH was adjusted using a Ph meter and tween 20 was added. To make 1 strength of PBS-Tween for washing, 200mls of the 10X stock was measured into measuring cylinder, distilled water was added to make 2000mls of wash solution. 1 liter of Conjugate Buffer 25mls of PBS without tween was added to 475 mls of distilled water containing 1g of PVP (Polyvinyl pyrrolidone) 0.1g of Albumin 250µl of Tween 20 was then added to make 500mls H 1 Liter of Coating Buffer P 9.6 1.59g of Sodium Carbonate 2.93g of Sodium hydrogen Carbonate H Were measured and diluted with distilled water, the P was adjusted to 9.6 H 1 Liter of Substrate Buffer P 9.8 97 mls Diethanolamine 800 mls Distilled Water The PH was adjusted using HCL and water added to make 1000mls. Buffers were stored at 4ºC and fresh buffers were prepared if the already prepared buffers were not used within one month. Buffers were check every two weeks for contaminants. 125 INFORMED CONSENT FORM My name is Mary Esien Kooffreh, I am a staff of the Dept of Genetics and Biotechnology, University of Calabar and currently a Ph.D student of the Dept of Zoology, University of Ibadan, Ibadan. We are carrying out a survey to find how many people are hypertensive or not, have certain elements in their blood that could make them hypertensive, you will be given a questionnaire to fill to help us find out if hypertension runs in your family or not and other factors that could make you develop hypertension. Please note that your name will not be used anywhere. A code number will be used for your questionnaire, and blood sample. The information you give will help us study more on hypertension and how this disease can be controlled effectively. Your weight, height and Blood pressure will be measured, about 3ml of blood will be collected from your arm, we will use this to look for certain elements in your blood that could make you develop hypertension and also check if you have HIV infection. You are free to turn down or refuse to participate in this survey. We will appreciate if you take part in all aspects of the survey as this will form baseline information for this area as not much work has been done in this region. The test is free and you do not have to pay any monies. We thank you for your co-operation. CONSENT: Now that the study has been explained to me and I understand the content of the study process I will be willing to take part in the programme. { } Hypertension { } HIV Your wish will be strictly adhered to. ________________________________ _____________________ Signature/Thumb print of participant/Date Signature Interviewer/Date ______________________________ Signature/Thumb print of witness/Date INFORMED CONSENT FORM (IBIBIO) Ami nkere Mary Esien Kooffreh. Ndo kiet ke otu mmon enama utom ke department Genetics ye Biotechnology, Ufok nwed ntaifiok ke Calabar. Nko idaham nka nwed ke ufok ke Ibadan man otodo enno ekamba nwed itoro mmo ekoto Ph.D ke Zoology. Nnyin idomo ukeme adi duno ndiono owo ifan edonoke nkonnkon iyip ye mmo mi idonoke. Iya uno fien nwed yak aboro nbime man otodo nnyin idiono mme nkonnkon iyip emi asanga ke ubon mfo ye mme nkpo afen ekemeke adi nam afo anie nkonnkon iyip. Ikpa ima adi neke nam afo adiono ke owo iyeme ayin nfo. Number ke idi dad inim idion ubana fien ye etok iyip nnyin isiogho fien ke ubok. Mme iboro afo noho aya wam nnyin adi neke mkpeep kpo mbana nkonnkon iyip, iyun iteme mmon edonoke enye nana ekpe kama idem ammo. 126 Iya udomo fien man idiono nana afo anion atre ye nana afo adopoke, inyun udomo fien ubok adi diono mme afomenie iden nkonnkon iyio ami. I ya inyun ise me afo me dono udono ado ekoto itiaita. Amekeme isin nkpo afo muyeme enam ye iyip mfo, ado, ikpa ukom fien tutu adieke afo yimeke adi wana ke efit se nyin inam. Nyim iyeme afo kpe akok nte kiet-O. Sosono nana anwam nyin adinam utom ami UNYIME: Sia ema tan utom ami anwana mien, ami mme nyime adi wana ke esit nyun nno mbufo iyip mi eben ese mme menie ( ) nkonnkon iyip; ( ) udono itiaita Se afo amek ke edi nam. ………………………………. …………………………. Nsin ubok/ usen afion Nsin ubok andibip mbime /usen afion ………………………………. Nsin ubok ntiense/usen afion INFORMED CONSENT FORM (EFIK) Eyinn mi edi Mary Esien Kooffreh. Ndi kiet ke out mbon emi enamde uton ke department Genetics ye Biotechnology, ufok nwed ntaifiok ke Calabar. Ndien idaha emi, nka nwed ke ufok nwed ntaifiok ke Ibadan man eno mi akamba nwed itoro emi mmo ekotde Ph.D ke Zoology. Nnyin iwana ndi dunnode ndiong owo ifang aemi edonnode nkonnkon iyip ye mmo emi mi donnoke. Iye inofi nwed oboro mbume man otodo nnyin idionno me nkonnkon iyip emi asnga ke ubon fo ye mme mkpo efen ekemede ndi nam fi eyene nkonkon iyip. Ikpi ma ndi neghede nnam fi ofiok ke owo iyomke eyinn fo. Number ke edi da inim idionno ibanga fi ye ekpri iyip nnyin isiode fi ke ubok. Mme iboro emi afo onode eye wam nnyin ndi neghede nkpep nkpo mbagha nkonnkon iyip, nyung mkpep mmo emi edonnode enye nte ekpe kamade idem mmo. Iye domo fi man idionno nte afo onionde tre ye nte afo odobide, iyung idomo fi ubok man idionno me afo me yene idem nkonnkon iyip. Iye iyunn ise me afo mo donno udonno odo ekotde itiaita, eme keme ndi sin mkpo mi afo mu magha yak enam ye iyip fo, edi ikpi kom fi etieti edieke afo yimede ndi buana ke kpukpuru nkpo nyin inamde. Nyin iyomke fi ekpe okwuk ndomo kiet-O. Sosongo ke ndi wam nyin inam utom emi. Mme yime: Sia ema ke tinn utom mmufo anwanga mi, me yime ndi buana ke esit nyung nno mbufo iyip mi eda ese me mmeyene: ( )nkonnkon iyip; ( ) udonno itiaita Se afo mekde ke edi nam. ………………………………… ………………………… Sin ubok nwed fo/ usen ofiong ubok nwed andibup mbume/usen ofiong 127 ………………………………... Ubok nwed ntiense/usen ofiong QUESTIONNAIRE Please fill in the boxes and tick right where applicable. CODE NO: Age { } Weight { } Sex { } Height { ) Ethnic Group { } BP { } Marital Status { } Systolic { } Diastolic { } EDUCATIONAL QUALIFICATION HIV STATUS: { } Primary Negative { } { } Secondary Positive { } { } University Don‟t know { } What is the level of your stress in your work place? High { } Low { } Moderate { } None { } Occupation: --------------------------------------------- Are you Hypertensive? { } Yes { } No If yes when were you first diagnosed? Is there anybody that has suffered from hypertension or stroke in your family before? { } Yes { } No If yes, please indicate. Brother/sister { } 128 Mother/father { } Uncle/Aunties { } Grandfather/mother { } If you are hypertensive, what type of hypertensive drugs are you taking------------------ --------- dosage ------------------------------ Do you smoke? { } Yes { } No How often do you take hot drinks. On a daily basis at weekends One glass { } { } One bottle { } { } Two or three bottles { } { ) More than three bottles { } { } Do you do any form of exercise daily like jogging { } Playing games { } Swimming { } Please mention any other form of exercise that you do ------------------------- Do you like plenty of salt in your food? { } Yes { } No How often do you go to fast food joint? Daily { } Weekends { } How often do you eat snacks for breakfast? { } for lunch { } dinner { } in between meals What types of snack do you like? Meat pies { } Egg rolls { } Cakes { } Sausage roll { } Bread { } T test for popn Group Statistics Std. Std. Error GROUPS N Mean Deviation Mean HT 1 612 1.6196 .07947 .00321 2 612 1.8754 6.76715 .27355 BMI 1 612 23.3201 5.83216 .23575 2 612 27.4856 5.80769 .23476 WT 1 612 65.4199 12.75368 .51554 2 612 70.6062 15.02097 .60719 SYS 1 612 1.1692E2 9.21780 .37261 2 612 1.6111E2 23.26299 .94035 DIAST 1 612 72.4804 8.36550 .33816 129 2 612 93.2516 13.76856 .55656 AGE 1 612 31.9216 10.40680 .42067 2 612 51.2778 13.75807 .55614 130 T test for ELISAS Group Statistics Std. Std. Error groups N Mean Deviation Mean Onehr 0 199 .1753 .06879 .00488 1 199 .1607 .04888 .00346 threehrs 0 199 .3806 .19552 .01386 1 199 .2605 .12421 .00880 overnight 0 199 .6633 .33251 .02357 1 199 .4949 .20958 .01486 Gene 0 199 2.8794 .34161 .02422 1 199 2.8894 .33004 .02340 Age 0 199 34.5628 10.27426 .72832 1 199 53.4523 14.14451 1.00268 Bmi 0 199 23.0556 6.51740 .46201 1 199 26.3190 5.78990 .41043 systolic 0 199 1.1566E2 9.63119 .68274 1 199 1.6307E2 23.87152 1.69221 diastolic 0 199 71.3970 8.50895 .60318 1 199 94.8744 13.59067 .96342 131 Independent Samples Test Levene's Test for Equality of Variances t-test for Equality of Means Std. 95% Confidence Sig. Mean Error Interval of the Sig (2- Differe Differe Difference F . t df tailed) nce nce Lower Upper HT Equal variances .07 3.156 -.935 1222 .350 -.25587 .27356 -.79258 .28084 assumed 6 Equal variances not 611.1 -.935 .350 -.25587 .27356 -.79311 .28137 assumed 69 BM Equal variances - - - .21 - I assumed 1.555 12.52 1222 .000 4.1655 .33270 3.5127 3 4.81826 0 2 9 Equal variances not - - - 1.222 - assumed 12.52 .000 4.1655 .33270 3.5127 E3 4.81826 0 2 9 WT Equal variances - - 19.33 .00 - - assumed 1222 .000 5.1862 .79653 3.6235 1 0 6.511 6.74899 7 6 Equal variances not - - - 1.191 - assumed .000 5.1862 .79653 3.6235 6.511 E3 6.74903 7 2 SY Equal variances - - - - 229.8 .00 1.0114 S assumed 43.68 1222 .000 44.184 46.1690 42.200 47 0 8 3 64 7 21 Equal variances not - - - - 798.2 1.0114 assumed 43.68 .000 44.184 46.1701 42.199 49 8 3 64 2 16 DI Equal variances - - - - 70.25 .00 AS assumed 31.89 1222 .000 20.771 .65124 22.0489 19.493 0 0 T 5 24 1 58 Equal variances not - - - - 1.008 assumed 31.89 .000 20.771 .65124 22.0491 19.493 E3 5 24 8 31 132 AG Equal variances - - - - 29.22 .00 E assumed 27.75 1222 .000 19.356 .69732 20.7242 17.988 9 0 8 21 8 14 Equal variances not - - - - 1.138 assumed 27.75 .000 19.356 .69732 20.7243 17.988 E3 8 21 8 04 133 Independent Samples Test Levene's Test for Equality of Variances t-test for Equality of Means 95% Confidence Interval of the Mean Difference F Sig. t df Sig. (2-tailed) Difference Std. Error Difference Lower Upper onehr Equal variances assumed .594 .441 2.445 396 .015 .01463 .00598 .00287 .02639 Equal variances not assumed 2.445 357.305 .015 .01463 .00598 .00286 .02639 threehr s Equal variances assumed 9.216 .003 7.314 396 .000 .12010 .01642 .08781 .15238 Equal variances not assumed 7.314 335.433 .000 .12010 .01642 .08780 .15240 overnig ht Equal variances assumed 19.229 .000 6.044 396 .000 .16840 .02786 .11363 .22318 Equal variances not assumed 6.044 333.874 .000 .16840 .02786 .11359 .22321 gene Equal variances assumed .343 .559 -.298 396 .765 -.01005 .03367 -.07625 .05615 Equal variances not assumed -.298 395.531 .765 -.01005 .03367 -.07625 .05615 age Equal variances assumed 11.527 .001 -15.242 396 .000 -18.88945 1.23928 -21.32584 -16.45306 Equal variances not assumed -15.242 361.440 .000 -18.88945 1.23928 -21.32655 -16.45234 bmi Equal variances assumed 2.297 .130 -5.281 396 .000 -3.26337 .61799 -4.47831 -2.04842 Equal variances not assumed -5.281 390.579 .000 -3.26337 .61799 -4.47836 -2.04837 systolic Equal variances assumed 95.678 .000 -25.977 396 .000 -47.40201 1.82474 -50.98941 -43.81461 Equal variances not assumed -25.977 260.797 .000 -47.40201 1.82474 -50.99512 -43.80890 diastolic Equal variances assumed 24.392 .000 -20.655 396 .000 -23.47739 1.13666 -25.71204 -21.24274 Equal variances not assumed -20.655 332.552 .000 -23.47739 1.13666 -25.71334 -21.24143 134 a Coefficients Standardized Unstandardized Coefficients Coefficients Correlations Collinearity Statistics Model B Std. Error Beta t Sig. Zero-order Partial Part Tolerance VIF 1 (Constant) 66.793 12.300 5.430 .000 AGT -2.224 2.215 -.033 -1.004 .316 -.074 -.041 -.033 .978 1.023 AGE .192 .061 .113 3.150 .002 .105 .128 .103 .828 1.208 SEX .679 1.763 .014 .385 .700 -.015 .016 .013 .800 1.250 ETHNIC .246 .304 .028 .811 .418 .117 .033 .027 .874 1.145 MARITAL 1.531 1.426 .037 1.074 .283 .086 .044 .035 .912 1.096 BMI -.192 .149 -.048 -1.290 .197 -.094 -.053 -.042 .778 1.286 DIASTOLIC .923 .057 .546 16.111 .000 .564 .552 .527 .932 1.073 EDUCATION -.858 .896 -.035 -.957 .339 -.139 -.039 -.031 .803 1.245 STRESS .146 .720 .007 .203 .839 -.106 .008 .007 .898 1.113 OCCUPATION -.190 .202 -.033 -.938 .349 -.103 -.038 -.031 .888 1.126 KNOWLEDGE 2.255 1.757 .044 1.283 .200 .014 .053 .042 .899 1.112 FAMHISTORY 1.602 .986 .054 1.625 .105 .054 .067 .053 .964 1.038 SMOKING 7.361 6.492 .038 1.134 .257 .070 .047 .037 .947 1.056 ALCOHOL -1.324 .993 -.045 -1.333 .183 -.077 -.055 -.044 .927 1.079 EXERCTYPE -.393 .607 -.022 -.647 .518 -.062 -.027 -.021 .943 1.060 SALT 1.906 2.094 .031 .911 .363 .056 .037 .030 .943 1.061 FASTFOOD -5.591 2.565 -.075 -2.179 .030 -.150 -.089 -.071 .913 1.095 SNACKUSE .220 .602 .012 .366 .714 .076 .015 .012 .949 1.053 a. Dependent Variable: SYSTOLIC 135 a Coefficients Standardized Unstandardized Coefficients Coefficients Correlations Collinearity Statistics Model B Std. Error Beta t Sig. Zero-order Partial Part Tolerance VIF 1 (Constant) 48.074 7.274 6.609 .000 AGT -.076 1.326 -.002 -.057 .954 -.047 -.002 -.002 .976 1.025 AGE -.144 .036 -.144 -3.989 .000 -.050 -.162 -.132 .836 1.196 SEX -1.533 1.053 -.054 -1.456 .146 -.054 -.060 -.048 .803 1.246 ETHNIC .191 .182 .037 1.051 .293 .121 .043 .035 .874 1.144 MARITAL .430 .854 .017 .504 .614 .036 .021 .017 .911 1.098 BMI .091 .089 .039 1.028 .304 -.040 .042 .034 .777 1.287 EDUCATION -.178 .536 -.012 -.332 .740 -.060 -.014 -.011 .802 1.247 STRESS -1.199 .428 -.097 -2.802 .005 -.144 -.114 -.093 .910 1.099 OCCUPATION -.171 .121 -.050 -1.417 .157 -.084 -.058 -.047 .890 1.124 KNOWLEDGE -1.495 1.051 -.050 -1.423 .155 -.023 -.058 -.047 .900 1.111 FAMHISTORY -.670 .591 -.038 -1.135 .257 -.001 -.047 -.038 .962 1.040 SMOKING 1.576 3.887 .014 .406 .685 .039 .017 .013 .945 1.058 ALCOHOL .397 .595 .023 .668 .504 -.027 .027 .022 .925 1.081 EXERCTYPE .141 .363 .013 .387 .699 -.010 .016 .013 .943 1.061 SALT .365 1.253 .010 .291 .771 .040 .012 .010 .941 1.062 FASTFOOD -.906 1.540 -.020 -.588 .557 -.109 -.024 -.019 .907 1.103 SNACKUSE .222 .360 .021 .617 .538 .061 .025 .020 .950 1.053 SYSTOLIC .330 .020 .558 16.111 .000 .564 .552 .533 .913 1.096 a. Dependent Variable: DIASTOLIC 136 a Coefficients Standardized Unstandardized Coefficients Coefficients Correlations Collinearity Statistics Model B Std. Error Beta t Sig. Zero-order Partial Part Tolerance VIF 1 (Constant) 82.217 6.324 13.000 .000 ATIR -2.110 3.231 -.023 -.653 .514 .006 -.027 -.022 .972 1.029 ACE .115 .478 .008 .242 .809 -.007 .010 .008 .989 1.011 AGT -.048 1.125 -.001 -.042 .966 .006 -.002 -.001 .971 1.030 AGE .076 .042 .086 1.790 .074 .169 .073 .061 .507 1.974 SEX -3.195 .694 -.172 -4.606 .000 -.206 -.186 -.157 .829 1.206 ETHNIC .162 .149 .038 1.089 .276 .002 .045 .037 .976 1.024 MARITAL -.871 .857 -.049 -1.017 .310 .059 -.042 -.035 .498 2.010 BMI .064 .056 .040 1.132 .258 .069 .046 .039 .917 1.091 DIASTOLIC .550 .039 .499 13.962 .000 .521 .498 .476 .909 1.100 EDU -.163 .415 -.015 -.392 .695 -.008 -.016 -.013 .790 1.266 STRESS -.028 .244 -.004 -.113 .910 .029 -.005 -.004 .867 1.153 OCCUPATION -.117 .082 -.053 -1.424 .155 -.012 -.058 -.049 .852 1.174 FAMHISTORY -.388 .384 -.036 -1.009 .313 -.032 -.041 -.034 .937 1.067 SMOKING -.193 1.930 -.004 -.100 .920 -.033 -.004 -.003 .926 1.079 ALCOHOL .240 .358 .024 .671 .503 .055 .028 .023 .922 1.085 EXERCISE -.086 .145 -.022 -.596 .552 .014 -.024 -.020 .866 1.155 SALT .470 .749 .022 .628 .530 .005 .026 .021 .972 1.029 FASTFOOD -.391 .727 -.020 -.538 .591 -.004 -.022 -.018 .876 1.142 SNACKUSE -.172 .251 -.024 -.683 .495 -.006 -.028 -.023 .964 1.037 a. Dependent Variable: SYSTOLIC 137 a Coefficients Standardized Unstandardized Coefficients Coefficients Correlations Collinearity Statistics Model B Std. Error Beta t Sig. Zero-order Partial Part Tolerance VIF 1 (Constant) 7.013 6.487 1.081 .280 ATIR 4.120 2.922 .049 1.410 .159 .050 .058 .048 .974 1.026 ACE -.262 .433 -.021 -.606 .545 -.029 -.025 -.021 .990 1.010 AGT .592 1.018 .020 .582 .561 .015 .024 .020 .971 1.030 AGE .100 .038 .125 2.623 .009 .222 .107 .089 .510 1.962 SEX .473 .639 .028 .740 .460 -.076 .030 .025 .801 1.248 ETHNIC -.264 .135 -.067 -1.960 .051 -.066 -.080 -.067 .981 1.020 MARITAL .708 .776 .044 .913 .362 .149 .037 .031 .497 2.010 BMI .085 .051 .060 1.680 .093 .114 .069 .057 .919 1.088 EDU .266 .375 .027 .709 .479 -.017 .029 .024 .791 1.265 STRESS .476 .220 .079 2.164 .031 .063 .089 .074 .874 1.144 OCCUPATION .021 .075 .010 .281 .779 .040 .012 .010 .849 1.178 FAMHISTORY .211 .348 .021 .606 .545 .008 .025 .021 .936 1.068 SMOKING -.407 1.747 -.008 -.233 .816 -.006 -.010 -.008 .926 1.079 ALCOHOL .154 .325 .017 .475 .635 .026 .020 .016 .921 1.085 EXERCISE .009 .131 .003 .071 .943 -.021 .003 .002 .865 1.156 SALT -.731 .678 -.037 -1.079 .281 -.050 -.044 -.037 .973 1.028 FASTFOOD .235 .658 .013 .357 .721 -.008 .015 .012 .875 1.142 SNACKUSE .408 .227 .062 1.795 .073 .050 .074 .061 .969 1.032 SYSTOLIC .451 .032 .497 13.962 .000 .521 .498 .475 .914 1.095 a. Dependent Variable: DIASTOLIC 138 PTSYSTOLIC Model Summaryb R Adjusted R Std. Error of Durbin- Model R Square Square the Estimate Watson 1 .604a .365 .346 18.81339 1.938 a. Predictors: (Constant), SNACKUSE, SEX, FAMHISTORY, AGT, OCCUPATION, STRESS, KNOWLEDGE, SMOKING, EXERCTYPE, MARITAL, DIASTOLIC, FASTFOOD, SALT, ALCOHOL, ETHNIC, EDUCATION, AGE, BMI b. Dependent Variable: SYSTOLIC PTDIASTOLIC Model Summaryb R Adjusted R Std. Error of Durbin- Model R Square Square the Estimate Watson 1 .593a .352 .332 11.25225 1.875 a. Predictors: (Constant), SYSTOLIC, KNOWLEDGE, SEX, FAMHISTORY, AGT, EXERCTYPE, OCCUPATION, SNACKUSE, STRESS, SMOKING, MARITAL, SALT, ALCOHOL, FASTFOOD, ETHNIC, EDUCATION, AGE, BMI b. Dependent Variable: DIASTOLIC CTSYSTOLIC Model Summaryb R Adjusted R Std. Error of Durbin- Model R Square Square the Estimate Watson 1 .559a .313 .291 7.76312 1.868 a. Predictors: (Constant), SNACKUSE, EXERCISE, ACE, BMI, ETHNIC, SALT, AGT, SMOKING, ATIR, OCCUPATION, ALCOHOL, DIASTOLIC, FASTFOOD, FAMHISTORY, STRESS, SEX, EDU, AGE, MARITAL b. Dependent Variable: SYSTOLIC 139 CTDIATOLIC Model Summaryb Adjusted R Std. Error of Durbin- Model R R Square Square the Estimate Watson 1 .559a .313 .291 7.76312 1.868 a. Predictors: (Constant), SNACKUSE, EXERCISE, ACE, BMI, ETHNIC, SALT, AGT, SMOKING, ATIR, OCCUPATION, ALCOHOL, DIASTOLIC, FASTFOOD, FAMHISTORY, STRESS, SEX, EDU, AGE, MARITAL b. Dependent Variable: SYSTOLIC 140 141 142 143 144 145