Association of angiotensin-converting enzyme gene insertion/deletion polymorphisms with risk of hypertension among the Ethiopian population

Introduction Although the pathophysiological mechanism of hypertension is not fully elucidated yet, a large number of pieces of evidence have shown that genetic alterations in the renin-angiotensin-aldosterone system play a central role. However, the association of insertion/deletion polymorphism of the angiotensin-converting enzyme (ACE) gene with essential hypertension is controversial yet, and there is a limited number of publications among the Ethiopian population. Therefore, this study aimed to determine the association of ACE gene I/D polymorphism with the risk of hypertension among essential hypertension patients at the University of Gondar Comprehensive Specialized Hospital, Gondar, Ethiopia. Materials and methods A case-control study was conducted from October 07, 2020, to June 02, 2021, among hypertensive patients and normotensive control groups at the University of Gondar Comprehensive Specialized Hospital. A structured questionnaire was used to collect socio-demographic data and anthropometric measurements. Five milliliters of blood were drawn from each of the randomly selected 64 hypertensive and 64 normotensive participants for molecular test analysis. Genetic polymorphism of the ACE gene was identified using polymerase chain reaction (PCR) and electrophoresis. Data analysis was done using SPSS version 25.0 software. The strength of association between the genotype and hypertension was estimated through the calculation of adjusted odds ratio and 95% confidence intervals using logistic regression. P-value < 0.05 was considered statistically significant. Result The distribution of DD genotypes and D allele of the ACE gene were 48.4% and 63% in essential hypertensive patients, respectively, while it were 29.7% and 42.2% in control subjects respectively. The ACE DD genotype (p-value = 0.005) and D allele (p-value = 0.001) were more frequent among hypertensive patients as compared to controls. Conclusion The present study found that the DD genotype and D allele of the ACE gene has had a strong association with a high risk of hypertension in the study population.


Introduction
Although the mechanism of hypertension pathophysiology is not fully elucidated yet, a large number of pieces of evidence have shown that genetic alterations in the reninangiotensin-aldosterone system play a central role. However, the association of insertion/deletion polymorphism of the angiotensin-converting enzyme (ACE) gene with essential hypertension is controversial yet, and there is a limited number of publications among the Ethiopian population. Therefore, this study aimed to determine the association of ACE gene I/D polymorphism with the risk of hypertension among essential hypertension patients at the University of Gondar Comprehensive Specialized Hospital, Gondar, Ethiopia.

Materials and methods
A case-control study was conducted from October 07, 2020, to June 02, 2021, among hypertensive patients and normotensive control groups at the University of Gondar Comprehensive Specialized Hospital. A structured questionnaire was used to collect socio-demographic data and anthropometric measurements. Five milliliters of blood were drawn from each of the randomly selected 64 hypertensive and 64 normotensive participants for molecular test analysis. Genetic polymorphism of the ACE gene was identified using polymerase chain reaction (PCR) and electrophoresis. Data analysis was done using SPSS version 25.0 software. The strength of association between the genotype and hypertension was estimated through the calculation of adjusted odds ratio and 95% confidence intervals using logistic regression. P-value < 0.05 was considered statistically significant.

Result
The distribution of DD genotypes and D allele of the ACE gene were 48.4% and 63% in essential hypertensive patients, respectively, while it were 29.7% and 42.2% in control subjects respectively. The ACE DD genotype and D allele were more frequent among hypertensive patients as compared to controls.

Conclusion
The present study found that the DD genotype and D allele of the ACE gene has had a strong association with a high risk of hypertension in the study population.    25.0 software. The strength of association between the genotype and hypertension was 27 estimated through the calculation of adjusted odds ratio and 95% confidence intervals using 28 logistic regression. P-value < 0.05 was considered statistically significant. 29 Result: The distribution of DD genotypes and D allele of the ACE gene were 48.4% and 63% 30 in essential hypertensive patients, respectively, while it were 29.7% and 42.2% in control 31 subjects respectively. The ACE DD genotype and D allele were more frequent among 32 hypertensive patients as compared to controls. 33

Conclusion:
The present study found that the DD genotype and D allele of the ACE gene has 34 had a strong association with a high risk of hypertension in the study population. 35 Keywords: Hypertension, ACE gene, Gene polymorphism, Genotype 36

INTRODUCTION 37
Hypertension is a state of elevated blood pressure which can be defined as either systolic blood 38 pressure at or above 130 mmHg and/or diastolic blood pressure at or above 80 mmHg (1). 39 Currently, hypertension is becoming one of the most common public health problems 40 worldwide. Globally, it is estimated that around 40% of adults are lived with hypertension with 41 great regional and residence variations, and the majority of them (46%) are found in Africa 42 (2). Nearly one billion people have hypertension; of these, two-thirds are in developing 43 countries (3). Besides, in 2015, 8.5 million deaths were associated with high blood pressure, 44 88% of which were in low-income and middle-income countries (4). The prevalence of high 45 BP is predicted to increase by 24% in developed nations and by 80% in developing regions 46 (5). Likewise, the incidence of hypertension is increasing alarmingly in various populations of 47 Ethiopia and other developing nations (2, 6, 7). In Ethiopia, the estimate from meta-analysis 48 and community-based studies revealed that the prevalence of hypertension ranged from 13% 49 to 35%, and factors such as socio-demographic, economic, biological, and behavioral 50 characteristics were found to be significantly associated with hypertension (2, 6-9). But there 51 are no genetic studies among Ethiopian hypertensive patients. 52 Numerous candidate genes have been implicated in susceptibility to essential hypertension 53 (10). In recent years, genes of the renin-angiotensin-aldosterone system (RAAS) have received 54 a good deal of attention (11-13). Thus, RAAS genes that encode for angiotensinogen (AGT), 55 angiotensin type 1 receptor (AT1R) and angiotensin-converting enzyme (ACE) have been 56 widely investigated in different ethnic populations (13). The ACE gene that encodes ACE is 57 one of them that has received a good deal of attention in recent studies. The key enzyme of this 58 system (ACE) catalyzes the production of angiotensin II, which acts as a strong vasoobliterant 59 and stimulates the secretion of aldosterone (10, 12, 14).

95
All essential hypertensive patients for cases and apparently healthy normotensive individuals 96 for controls who visit the University of Gondar Comprehensive Specialized Hospital were the 97 source population of the study. All essential hypertensive patients for cases who were newly 98 diagnosed and at follow-up at the time of the study period and apparently healthy normotensive 99 individuals for controls attending the University of Gondar Comprehensive Specialized 100 Hospital during the study period were the study population of the study. For both cases and 101 controls, individuals with age less than 18 years and greater than 65 years on both genders 102 and/or if they are clinically confirmed to have comorbidity such as tuberculosis, diabetes 103 mellitus, liver disease, pregnancy hypertension, renal disease, inflammatory disease, thyroid 104 disease, and all others with secondary hypertension were excluded. Pregnant or postpartum 105 period women were also excluded from the study. 106 5 | P a g e

107
The sample size was calculated using G* power version 3.1.9.4 software by selecting an 108 independent t-test (28). It is calculated by considering alpha= 0.05, power (1-β) = 0.8 (80%), 109 effect size (d) = 0.5 and allocation ratio N2/N1 =1, then the total sample size became 128. 110 Therefore, by a 1:1 case-control ratio of 64 hypertensive cases and 64 age sex-matched healthy 111 controls were recruited as study participants using a simple random sampling technique. Waist and hip circumference were measured using a standard non-stretchable measuring tape. 131 The waist circumference was measured around the abdomen at the level midway (smallest 132 6 | P a g e horizontal girth) between the lowest rib margin and the iliac crest at the end of expiration. The 133 hip circumference was also measured at the levels of widest diameters around the buttocks (at 134 the broadest part of the hips). Then, waist to hip ratio (WHR) was calculated by dividing waist 135 circumference by hip circumference. According to WHO (2019), the cut-off point considered 136 for waist circumference (WC) was >88cm for females and >102cm for males to define 137 overweight, the cut-off taken for waist to hip ratio was >0.85 for females and >0.90 for males 138 to define overweight. 139 Blood pressure was measured after a minimum rest of 5 minutes, or 30 minutes for those who 140 had drunk hot drinks such as coffee, with a sphygmomanometer at the midpoint of the left arm 141 in the sitting position with arm support. The blood pressure was measured twice with an 142 interval of 5 minutes, and the average value was taken as the true value. The cut-off points for 143 elevated BP were SBP of 130mmHg or above and DBP of 80mmHg or above (1). 144

145
The collection of blood samples was done by experienced medical laboratory technologists. A 146 volume of 5ml of blood was collected using EDTA coated tube by certified health care 147 professionals in the Hospital from each participant. Then, the blood samples were kept in a -148 4 o C refrigerator for genetic analysis through the salting out method. 149 Genomic DNA isolation: DNA extraction was done using the non-enzymatic salting-out 150 method (29), by taking 300µl of EDTA anti-coagulated blood of both patients and healthy 151 controls and transferring it to a sterilized 1.5ml Eppendorf tube. The red blood cells (RBC) 152 lysis buffer solution was used to lysis and remove RBCs. Similarly, white blood cells were 153 lysed using a nuclear lysis buffer solution. Then, a highly concentrated salt (6M NaCl) was 154 added to precipitate and remove proteins. The DNA was precipitated by chilling using 155 isopropanol followed by washing with 70% ice-cold ethanol. Then, genomic DNA was 156 dissolved with Tris-EDTA buffer (TE) and stored at -21ºC till use. The quality of isolated 157 genomic DNA was confirmed by using 1.5% agarose gel electrophoresis or by measuring its 158 7 | P a g e absorbance ratio at 260/280nm (Figure 2). Then, the samples were genotyped for ACE gene 159 (I/D) polymorphism using sets of primers and appropriate PCR conditions (Annex 1 and 3). The PCR amplification was set with an initial denaturation and activation of an enzyme at 95°C 167 for two minutes. Then, the DNA was amplified for 30 cycles. The cycle steps were denaturation 168 at 94°C for 30 seconds, annealing at 58°C for 30 seconds and extension at 72°C for 45 seconds 169 followed by a final extension at 72°C for 9 minutes. Finally, the PCR product was held at 4°C 170 until it becomes analyzed by agarose gel electrophoresis.  (table 1). 235 respectively. The BMI, WHR, SBP, and DBP were significantly higher in hypertensive 244 patients than in controls (p-value < 0.001). On the other hand, age (p-value=0.062) was not 245 significantly different between the study groups ( Table 2). 246

ACE genotype and allele distribution in HTN patients and healthy
The genomic DNA was isolated and its quality was confirmed by using agarose gel 253 electrophoresis (Figure 2). Then, the qualified genomic DNA samples were amplified and 254 genotyped through electrophoresis to identify the type of polymorphism by using the size of 255 base pairs of the bands formed. Then, the DNA samples were classed as II (490bp band), DD 256 (190bp band), and ID (both 490bp and 190bp band) (Figure 3). 257 The ACE genotypes frequency distribution in hypertensive patients and healthy control is 258 given in figure 4 and   implies that the DD genotype and D allele might increase the susceptibility of getting 286 hypertension among the participants and may also contribute to the relatively high prevalence 287 of HTN in the Ethiopian population. This substantial link of the DD genotype and the D allele 288 with increased risk of hypertension could be attributed to the deletion of a 287-bp non-coding 289 region in the 16th intron of the ACE gene, which would result in increased ACE gene 290 transcription and ACE activity. The higher levels of ACE convert angiotensin-I to angiotensin-291 II, a potent vasoconstrictor; and also inactivate bradykinin, a potent vasodilator (15, 17). High 292 angiotensin-II levels can affect artery musculature, increase peripheral resistance, and raise 293 blood pressure. It has also direct sodium-retaining effects through increasing the activity of the 294 Na + /H + exchanger and Na + /K + ATPase in the proximal tubule, the Na + /K + /2Cl − transport in the 295 loop of Henley, and multiple ion transporters in the distal nephron and collecting tubules. 296 Angiotensin II also causes the production of aldosterone from the adrenal glands, which 297 stimulates the epithelial cells of the kidneys to enhance salt and water reabsorption, resulting 298 in increased blood volume and blood pressure, and hypertension (5, 20). 299 In agreement with the present study, many reports worldwide disclosed this link between ACE 300 The BMI, WHR, SBP, and DBP were significantly higher in hypertensive patients than in 316 controls (p-value < 0.001). The present study found that hypertensive patients had significantly 317 higher BMI and WHR than normal controls. It might be because there is an association between 318 obesity and higher blood pressure which may cause obesity-induced hypertension through the 319 mechanism of insulin resistance, sodium retention, increased sympathetic nervous system 320 activity, activation of renin-angiotensin-aldosterone, and altered vascular function (38, 39). 321 This finding was in line with the findings from Bangladesh (40), China (14), and South Korea 322 (11), while it was inconsistent with the finding of a study conducted in southern India (15) and 323 Egypt (5). The inconsistency could be due to the variation in the ethnicity and socio-324 demographic condition of the study subjects. The present study found that hypertensive 325 patients had significantly higher SBP and DBP than normal controls. This finding is in line 326 with the study conducted in southern India (15), Bangladesh (40), China (14), and South Korea 327 (11). However, it is inconsistent with the study conducted in Egypt that reported there was no 328 significant difference in SBP and DBP among cases and controls (41). This discrepancy could 329